"You have a rodent infestation, shall I terminate?"
The Autobots attempt at 'hiding' in Sam's garden, leaving him with several large vehicles on his lawn.
Sam: This is not hiding! This is not hiding! This is my backyard, not a truck stop!
"This is my private area, my place of zen and peace...SHUT UP GRAMMA!!", followed by, "GRAMMA DRINK YOUR PRUNE JUICE!!"
Pretty much any scene with Glen in it, but especially the FBI interrogation room:
Glen: SHE DID IT! SHE DID IT! SHE'S THE ONE YOU WANT! I WAS JUST SITTING AT HOME WITH MY COUSIN WATCHIN' CARTOONS AND PLAYIN' VIDEO GAMES, AND SHE CAME IN AND... Maggie: Glen, you freak! Glen: DON'T TALK TO ME, DON'T TALK TO ME CRIMINAL! I AIN'T GOIN' TO JAIL FOR NOBODY!
Glen's cousin, especially when he tries to out run the FBI agents.
Glen's Cousin: (screams while running) I'M JUST A COUSIN! I'M JUST A COUSIN!
The real humor came after this line, where Mikaela finally comes out and says that Sam was with her. While Sam's mom tries to talk to her; Sam's dad gets this big shit-eating grin on his face and high-fives Sam in the background.
Made funnier by the fact that Julie White ad-libbed that line. The shocked confusion on Sam and Ron's faces and their spluttering responses? Genuine.
When Lennox's team are battling Scorponok outside a small desert villiage, Lennox attempts to contact the Pentagon. First, he needs to find a phone. When he finds someone with one, we get this jewel:
Lennox:(Making a gesture with his hand that clearly mimics him holding a phone to his head) Do you have a telephone? A telephone?
Later on, Lennox finally gets connected, but a bored Indian operator is hassling him because it is an international call and the Indian man does not buy Lennox's angry complaints about how he's in the middle of a battle and needs help immediately. The Indian man blandly insists that he needs a credit card to pay for the call.
Lennox: This is an emergency Pentagon call! The Pentagon, do you understand-? [window behind him blows out] NO, I DON'T HAVE A CREDIT CARD!!
International Operator:[bored] Sir, the attitude isn't going to speed things up any bit at all. I'm going to ask you to speak into the mouthpiece very clearly.
Capt. Lennox: I'm in the middle of a WAR! This is friggin' ridiculous!! [Runs over to Epps] I need a credit card! Epps, where's your wallet?
Tech Sgt. Epps: Pocket!!
Lennox: Which pocket?!
Epps: MY BACK POCKET!!!
Lennox: You've got ten back pockets!!!
Epps: LEFT CHEEK!!! LEFT CHEEK!!! LEFT CHEEK!!!
Lennox:[picks up card and continues talking] Okay, it's a Visa.
International Operator: Also, sir, have you heard about our Premium Plus full-service call package?
Lennox: NO, I DON'T WANT A PREMIUM PACKAGE!!! Epps, Pentagon!
The whole thing was based on stories that the film's military advisor told Michael Bay about real soldiers who went through almost identical issues when trying to contact their superiors at the Pentagon while in the middle of intense firefights.
Honestly, if some of the above entries weren't enough to tip you off, the scene where the Autobots are hiding from Sam's parents as he attempts to find his grandfather's glasses is absolutely priceless. First there's Sam looking back with a brief but very real look of utter horror when he sees Optimus in his normal form standing up and walking towards his house, followed by him coming up with increasingly ridiculous conversational topics to keep his father out of the back yard. When the Autobots come over, they wreak havoc on Sam's back yard. Then, when they suddenly realize that they should hide, they all transform. In the middle of Sam's back yard.
Hilarity Ensues as the Autobots accidentally convince the Witwicky family that they're caught in the middle of an earthquake. Sam's father looks outside and the Autobots are all seen hiding in the most ridiculous of places: Bumblebee has somehow wedged himself under the awning and the patio, Ratchet and Ironhide are kneeling between the windows, Jazz has found a location similar to Bumblebee's, except in the living room, and Optimus is so damn big that he can only press himself up against the house and avoid the windows. When he attempts to move, the camera pans to Sam's father as he's in the house. Then there's when Optimus comes up against the window and sees Sam's father. He goes Oh Crap and suddenly moves away, out of sight. There's a tremor and Sam's father leaps into the bath tub, shouting that there's been another earthquake, and how much he hates bathtubs.
Fridge Logic: Ron Witwicky is the head of the local neighborhood watch. He obviously sucks at his job.
Plus when Sector 7 show up, one of the things they do first is rip up Judy's flowers. She grabs abaseball batto beat the crap out of them. Sadly, she's stopped by Agent Simmons.
An exchange early on between Sam and two frat boys, after Bumblebee illegally parks on the college campus.
Sam: No, seriously dude, this car belongs to my friend over there. We were looking to see if we could get you a tighter shirt. Frat Boy 1:(wearing said tight shirt) THERE ARE NO TIGHTER SHIRTS. Frat Boy 2: WE CHECKED.
"You know who my father was? A wheel! The FIRST wheel! And you know what he transformed into? NOTHING! But he did so with honor! Dignity, dammit!"
"STARSCREAM, I'M HOME!" Someone else said it best: Domestic Abuse is funny when it involves giant robots.
"It's an ass-whuppin', it's supposed to hurt!"
"THIS IS COMBAT, MAN! WHAZZWRONG WIT'CHU?" *smacks Mudflap*
Jetfire: What sort of hideous mausoleum is this? Answer me, pawns and knaves! Show yourselves, or suffer my infinite wrath! You little spinal-cord-based organisms...! (bangs his head on a satellite, which drops, closely missing Sam) Jetfire: Oh, bugger it!
Sam trying to persuade Megatron not to kill him... with the exact same speech he tried to use to persuade his girlfriend not be be mad at him earlier in the movie.
Simmons telling Leo how not every adventure ends in success.
Sometimes you get to the end of the rainbow and the leprechaun went and booby-trapped it!
What am listening to you for? You live with your mother!
The entire scene where Lennox and his team ditch major Jerkass Galloway in the middle of some random Egyptian desert by faking some sort of a malfunction and convincing him that he needs to parachute out of the aircraft.
This is made even funnier when Galloway accidentally activates his parachute while still in the aircraft and Lennox yells: "Not now! We're on the plane, dumbass!" and Galloway is dragged out.
Earlier in the film, when Lennox informs General Morshower that Jerk Ass Galloway has been appointed liaison for the Autobots, he finishes with an eye roll.
During the debriefing with General Morshower, when Galloway starts berating Optimus that they didn't agree to share the Autobot weapon technology, this exchange takes place:
Epps: We've shed blood, sweat and precious metal together.
Galloway: Soldier, you're paid to shoot, not talk.
Epps (in an undertone): Don't tempt me.
The entire scene of the arrival to Egypt. "This'd be a perfect moment... except you landed on my testicles."
The entire sequence in Simmons' mother's butcher shop.
Mikaela: (sarcastically) You live with your mama?
Simmons: No, my mama lives with me. There's a difference.
When Laserbeak throws Wang out of the building he drops past the window where Bruce is having a meeting. His reaction is . . . hilarious
The whole exchange afterwards, in which Sam is ordered to clean up Jerry's body, is a brilliant Black Comedy moment.
Ironhide thumping the top of Optimus's cab to get him to transform while he is understandably angry after learning that they had been lied to. "Prime! Make something of yourself!" And then either Ironhide or one of the other Autobots going, "Oh yeah, he's pissed."
Mearing succinctly explaining why the audience hasn't seen the Wreckers up to now:
"We don't let them get off the base much because they're assholes."
When Simmons tries to get into the Russian bar:
Simmons: Dasvidania! Bouncer: That means "Goodbye." [slams peephole]
Anything to do with Sam's Datsun. "It reminds me of Bumblebee... if Bumblebee were a sad piece of shit."
Sam freaking out when a security guard at NEST headquarters bangs on the roof of the Datsun "DO NOT TOUCH MY CAR; IT'S A COLLECTOR'S ITEM!"
Sam's parents giving him a hard time about being unemployed. "So your car has a job?"
Optimus flies in to destroy the Driller, but gets blasted by Shockwave, thrown through a building and caught in construction wire on the other side. The great Optimus struggles feebly before giving up and yelling for the Wreckers to get him down. 15 minutes of Sam and the soldiers running around and getting shot at, and we cut back to see the Wreckers still haven't gotten him down yet and Prime is clearly starting to lose his patience.
Wheelie mouthing off to the NEST troopers in D.C. after Bumblebee shows up to vouch for Sam.
"Enjoy your next job at 7-11, dickhead."
Dylan referring to Megatron as "such a dick!" after Megatron "dismisses" him. Good thing Megatron didn't hear that.
When Epps is explaining his new job at keeping the peace between the Wreckers and the NASA techs, Roadbuster yells out in background that he's useless at it.
One of the Wreckers calls a human a "Nancy Wanker!" when they are first introduced.
Sam trying to hide the fact that Dylan put a Decepticon bug on him while talking to Mearing. The living contraption sends random nervous jerks through Sam, who ends up pulling a roll over two desks and come to a stop next to Mearing, who never even notices.
Hell, he wasn't trying to hide the fact he's got a Decepticon bug on him, he's trying to clue everyone else in that he has one without it killing him. Every random nervous jerk was the bug getting pissed off and punishing Sam for his hilarious attempts at blowing its cover.