Kaidan: If things don't go well, I want you to know - I've enjoyed serving under you.
Shepard: Kaidan, I don't think I've had the pleasure of you serving under me yet.
Kaidan: Ouch, I walked right into that one.
If you mod a save file to romance Kaidan as a male Shepard, this is one of the lines that was recorded before the option was cut, so the dialog plays out in its entirety. Mark Meer's delivery isn't quite as tongue-in-cheek, so he comes off as Straight Gay while Kaidan's lines are still delivered the same, like he's talking to a woman.
Ashley: Wow. Gives "down and give me twenty" a whole new meaning.
Shepard: You still sassin' me soldier? I think you need some more physical training.
Also, if you have Wrex with you when you first arrive on Noveria, and you have the confrontation with the dock guards.
Shepard: Easy. We don't want to start a fight.
Wrex: .....Why not?
Tali's reaction is just as good: "I'll be... right behind you. That armor is in good condition?"
And then there's the conversation with Lorik Qui'in, hilarious no matter who you bring with you: Kaidan's remark about asari and extranet fetish sites, Ashley's ironic Take That against the video game industry, Qui'in calmly asking Shepard to try not to get blood on his office carpets, and Wrex telling Qui'in that if the Executive Board doesn't support him, "you should eat them."
The fetish site thing is especially funny considering his earlier claim that Liara is "not to his taste", and he doesn't have much interest in "alien culture". Suuuuure.
He says that about Liara to Fem!Shep, after she asks if he finds their newest crewmember attractive. When talking to M!Shep the conversation goes along these lines:
M!Shep: Just remember that I saw her first.
Kaidan: Only by a few seconds, sir.
His comments regarding Chora's Den, filled with asari strippers:
I can see why this place is so popular. It's got quite the... view.
Though Ashley's reaction is much sillier:
"Hey, LT, put your tongue back in your mouth before you trip on it."
Lorik Qui'in has evidence that Administrator Anoleis is crooked and has Shepard retrieve the evidence. If you ask him to testify instead of using it for blackmail, he will refuse. You can convince him... OR you can just give the evidence to Anoleis instead (ruining Qui'in's career, and possibly his life) simply to backstab him. Upon telling Qui'in (who is naturally in shock), he asks why. The Renegade response?
Krogan Commander: Oh, good, I really need to kill somebody!
Trying to make small talk with the krogan bounty hunter Inamorda on Noveria.
Shepard: What's that you're drinking there?
Inamorda: Human blood! With cinnamon!
And if you negotiate a higher payment out of him and then turn him down...
Inamorda: Probably couldn't figure out how much more you'd make. (to himself) Stupid base-ten math. That's what they get for having extra fingers.
The elevator music. Makes the otherwise-boring elevator sequences pretty damn funny.
Speaking of the elevators, nearly everyone's elevator conversations on the Citadel. Bonus points for the mention of Francis Kitt's production of Hamlet with elcor actors. Becomes even funnier in Mass Effect 2, when you learn that this production of Hamlet is a riveting FOURTEEN HOURS long.
The much-maligned elevator rides are, at least, good for comedy:
Wrex: Hey, Tali. Your people created the geth, ever talk about it?
Tali: Do the krogan talk about starting a stupid war that got your species sterilized?
Wrex:All the time.
Liara: Our travels now are somewhat different from my normal excavations. I would prefer lengthier studies, and fewer explosions.
Tali: Yes. Most of the technology that I hoped to send back to the Flotilla has subsequently attempted to kill us.
Wrex: It's good for you. A nice explosion now and then keeps the mind sharp.
Ashley: I think you speak for scientists everywhere, Liara.
Wrex, on killing krogans despite the genophage: "Anyone who fights us is either stupid or on Saren's payroll. Killing the latter is business. Killing the former is a favor to the universe."
And when first talking to him on the Normandy: "There was this one time the turians almost wiped out our entire race. That was fun." It's the delivery that makes it hilarious.
Kaidan: I haven't spent much time with krogan before, Wrex. I have to say, you're not what I expected.
Wrex: Right. Because you humans have a wide range of cultures and attitudes, but krogan all think and act exactly alike.
Kaidan: Whoa. I-I didn't mean... Look, just forget I said anything.
All of Wrex's lines are pretty much made of hilarity. It's the way he delivers them. Everything is said in this completely deadpan voice where you can't quite tell if he's joking or not at first. He usually is, though—he probably does it on purpose because he thinks it's funny to watch other species squirm while they try to wriggle their way out of getting mauled by an angry krogan. It's just so funny that the oldest, grumpiest member of your squad is also the designated Deadpan Snarker.
Joker may be quite adept at being the Deadpan Snarker, but Wrex truly elevates this to an art form.
If you take Wrex on the mission to save Liara, when the cave-in starts and you go up the elevator to That One Boss, Wrex utters the immortal words:
Garrus: Some Krogan believe testicle transplants will increase their virility. Counteract the effects of the Genophage. It doesn't work but that doesn't stop them from buying. They'll pay up to 10,000 credits each. That's 40,000 for a full set *Beat* Somebody's making a killing out there.
Which gets referenced in the second game when Renegade Shepard, during Thane's loyalty mission, can threaten to cut Elias Kelham's balls off and sell them to a krogan.
If you didn't get the codes to start the Neutron Purge while on Noveria, one of Shepard's lines is "Sic Semper *cough*", in a shout out to Army of Darkness's "Klaatu...Barada...*mumble*" The VI will take a slight pause before denying access.
The party responses to Mira's explanation of how dangerous the neutron purge is are also good for a laugh. Wrex argues with it, claiming it would be like a walk in the sun for a krogan (whereas Mira claims that it's lethal to all lifeforms). If Liara and Ashley or Kaidan are with you, Liara tells Shepard the importance of reading the directions on any controls s/he uses, prompting Ashley/Kaidan to remark that it's always a good idea to RTFM, to Liara's confusion. If Tali is with you she will provide the immortal line:
Makes you wonder if her name is entirely coincidental. If it was, they probably noticed half way in and just ran with it.
When playing as an Adept, use the Lift power to fling a charging krogan into the air. It turns out that the game incorporated Newton's first law into the game, and the krogan will float up while still moving forward, drifting over Shepard's head while flailing uselessly. The kicker? The path of the krogan's drift can end up taking him over a railing.
A similarly hilarious result can be obtained with a combination of Lift, a sniper rifle, exploding rounds and insanely good timing. The sight of a burning krogan hurtling off toward the horizon makes it all worthwhile.
Sir Isaac Newton is the deadliest son of a bitch in space, after all.
Wrex's tone makes him sound surprised that she even asked the question, in the "you don't?" sense.
If you have Wrex and Kaidan, the conversation ends up going something like this.
Wrex: Who would win in a fight between you and Shepard?
Kaidan: What? Commander Shepard is my superior officer. I can't see us ever having to fight.
Wrex: You can't? That's why Shepard is your superior officer. And that's why... Shepard would win.
If one thinks about this, this also means that Wrex, one of the most badass individuals in the universe, believes Shepard could kick his butt most days since Wrex is willing to let Shepard order him around most of the time. He's not wrong. The standoff in ME3 proves that.
In a more lighthearted take on the above conversation, the Citadel DLC has a great Call Back to that scene, seeing as how you're fighting a clone of Shepard.
That entire scene with the hanar and the turian can come across as rather funny due to the fact that the turian officer just sounds so exasperated, like he's been putting up with this kind of crap all day and just wants to go home to take a nap.
Then there's this gem:
Asari Councilor: Do not cut me off again, Commander. I fail to find it amusing." Shepard: Whoops.
Also if you keep cutting off the Council, this happens.
Ashley trying to romance Shepard with poetry can lead to this reply.
"As I recall the captain dies in that poem."
That line is even funnier or more tragic in hindsight.
Ashley's conversation with male Shepard, after using Paragon to persuade Jeong into saving the colonists.
Shepard: I just pretend I have my head up my ass and don't care about anything except money.
Ashley: Yes, sir. Unless you ask me to kiss a superior officer. This would violate Alliance regulations on fraternization. At which point, I would refuse your request and relieve you of your command. ...Sir.
Liara: How humans tolerate such literal-minded VIs is beyond me...
Ashley's comments on Chora's Den is always good for a chuckle.
"A million light years from where humanity began and we walk into a bar filled with men drooling over half naked women shaking their asses on a stage. I can't decide if that's funny or sad."
Kaidan: What, you don't think they're here because of the food?
Appointing Captain Anderson as the human representative in the Council:
Ambassador Udina:Him? You must be joking. Anderson prefers his fists to do the talking.
Captain Anderson: Only with you, Ambassador. Only with you.
This is extra funny if you told Anderson to free the Normandy from Udina's office, where he does exactly that.
There's a hilarious bit in the Upper Markets on the Citadel. A human customer keeps attempting to make a return to a turian salesman. Their back and forth is made even funnier when another (turian) customer's return is accepted much more easily. (He has a proof of purchase, sir.)
If you follow it to the end he finds the receipt and gets a refund with no trouble. Until it continues in the sequel, of course...
Talking to Ashley about Eden Prime:
Shepard: The geth are perfect ambushers. They don't move, they don't sleep, they don't even breathe.
Ashley: Sir, they have flashlight heads!
Lorik Qui'in remarking on a human turn of phrase:
Shepard: (When finishing up a conversation with him) I should let you go.
Qui'in: "Let me go"? Do humans consider conversation a form of imprisonment? Maybe that is why so few are willing to sit and talk.
Ashley's legendary snark moment before you pick a second team leader on Virmire:
Ashley: Commander, we'll need your help with setting up the nuke. I'll go with the salarians.
Kaidan: With all due respect, gunnery chief, it's not your place to decide.
Made even better - or tragic - when Shepard subsequently says "with all due respect" to the Council during the argument that leads to the Normandy's lockdown. On a much lighter note again; Shepard seems to take Ashley's interpretation of the phrase every other times he/she says it.
Bringing Garrus to the Rogue AI assignment on the Citadel.
AI: Probability of detection 100%. Initiating self-destruct protocol.
Garrus: That could be a problem.
Bring Wrex and Liara, and Liara comments they should be able to just shut the machine down. Then the AI announces its self-destruct protocol.
Wrex: Or not.
Shepard seeing Species 37 a.k.a. the Thorian for the first time.
Alternatively, Shepard's weary and frustrated renegade response:
Shepard: Nothing's ever simple is it?
All three of the possible responses are pretty funny; the Paragon reaction is a humorously understated, "This could be... problematic."
Garrus's reaction is even better.
Garrus: This wasn't covered in my training manuals.
Tali: Now we should just be able to... to... Keelah, what is that?!?
In a minor sidequest on the Citadel, Shepard can either help the salarian Schells develop a device for cheating at space slot machines, or give the prototype device to the casino's owner and then go back and tell Schells about it. His flabbergasted reaction is already pretty funny, but when he asks what he's supposed to do now, Shepard's companions break out the snark:
Kaidan: Get a job?
Schells: You're no help at all!
Wrex and Kaidan tend to be a great combination for hilarity value. While on Noveria, when the rachni start banging around but before they're actually revealed:
After disabling the first torch in Bring Down the Sky, Simon Atwell mentions that one of the other torches is surrounded by a proximity minefield. Renegade Shepard's response?
Shepard: Could I requisition a cow to move ahead of me?
The Shifty Space Cow:
I love the history of the Shifty Cow easter egg. When the character artists designed the space cow with two extra arms, Preston, our lead designer, was a little creeped out. His comment was "You can't trust any animal that can milk itself." Those extra little hands look so... grabby. So Preston came up with the idea of Shifty Cow. Turn your back on him, and those creepy little hands are going to go to work. Kiss those credits goodbye.
The dialogue and delivery by the drugged scientists during the UNC: Besieged Base side mission can be hilarious.
Can you feel me growing!?I'm going to break it all!
If you bring Ashley with you after landing on the freezing cold world of Noveria and talk to her outside the ship, she gives the following good advice:
Ashley: Nobody lick any poles.
Admiral Hackett sends a Renegade Shepard to negotiate with a warlord who's trying to extort the Alliance. Shepard's reaction to getting this mission is basically, "You want me, of all people, to negotiate?" Darius makes several outrageous demands, and if you balk at them, you're forced to kill him. Later, reporting to Hackett, Shepard says, "You wanted me to kill him, didn't you?" Hackett is hilariously momentarily speechless before he says that the Alliance can't sanction assassination, but since you're a Spectre, there's nothing they can do.
Take Garrus with you when you turn in the Asari Diplomacy Assignment.
Nassana: "I am sorry I misled you. We have trust issues in our family."
The same thing can happen with any enemies on Insanity difficulty with the Mako's cannon, especially if they have Immunity active. Watching a random Mook go flying after taking a cannon shell to the face, only to land a couple hundred feet away, get up, and start shooting ineffectually at you, and then send flying/tumbling again, can get pretty fun pretty quickly.