The banter between Desmond and Lucy in the beginning.
Lucy: If I pulled all the levers, what would be left for you to do?
Lucy: Nice try.
When an Assassin recruit takes down Malfatto (aka the Doctor multiplayer character) in the main storyline, Ezio does the typical "Requiescat in pace" thing, and moves his hand as if to close the target's eyes (as he does with other targets). But since the Doctor wears a mask, Ezio just kinda hovers his hand over the beak, like he doesn't know what to do, before just leaving him.
Oddly enough, if you later complete the "Templar Agent" mission to kill Gaspar de la Croix (aka the Engineer multiplayer character), Ezio does do the "close eyes" motion with him despite de la Croix wearing glasses.
Examining Altair's statue in the present for the first time leads to this exchange.
Desmond: (In a stereotypical "Joisey" accent) Ey, wassa-matta-you AltaÔr?
Which then sparks an argument between Desmond, Lucy, and Rebecca about not working hard enough. After they're done fighting...
Shaun: Anyone up for some re-conciliatory yoga? Anyone?
Pretty much anything with Shaun and Rebecca.
Shaun: Bloody Third World country!
Rebecca: You're talking about Italy?
Shaun: No, just Europe in general.
Rebecca: I didn't know England was a Third World country.
Shaun: England isn't in Europe, Rebecca. We tolerate Europe. Like a person tolerates herpes.
Including Shaun Hastings, Dirty Yoghurt Thief.
Email from Rebecca: Shaun ate both of them. I know because he asked me if I wanted one.
Email from Lucy: Thanks Becca. Shaun, you're on clean-up for the next week.
Email from Shaun: This just in: Rebecca is a Templar.
A funny email from Shaun regarding his run-in with a large Italian guy:
Email from Lucy: You're still going in when it's your turn. If you need me to hold your hand and protect you from the big man just ask.
Email from Shaun: Fine. But when the brute eats my pancreas you'll be without a lead data manager...
When you get back from killing the Banker, and approach the Rosa in Fiore, you receive the next mission, stating that there's trouble inside. There are a bunch of crying courtesans outside, saying that the guards followed the one who took a money chest back. Ezio/the player enter the building, expecting a Player Punch since Ezio's mother and sister are inside... only to find a bunch of dead guards, surrounding a knife-wielding Claudia.
When Bartolomeo and Ezio are discussing the situation with the French.
Bartolomeo: We have them in retreat.
(a gunshot whizzes past them)
Ezio: ...They seem to be getting closer.
Bartolomeo: The situation is under control.
Mercenary: Close the gates!
Bartolomeo: Bene.note Alright. So maybe I could use a little help.
Also this line directed at Octavien de Valois:
Bartolomeo: You steal a man's wife and hide inside a fortress?! Nothing hangs between your thighs! In fact, there is a hole there so deep it reaches INTO THE MALEDETTO INFERNO! note Fucking underworld!
Bartolomeo: *patrolling the barracks. He draws Bianca as Ezio approaches* Who goes there?! Ezio: Salvenote Hello to you, too. Bartolomeo: Ezio! *sheathes his sword* I was expecting my wife.
Ezio: Somehow that does not surprise me.
During a Templar Agent side mission where you have to take down Gaspar de la Croix (aka the Engineer in multi-player), Ezio says it should be relatively easy to overwhelm a single gunman right before a bullet whizzes directly by his head. His shocked reaction sells it: "Merda..."note Shit...
After the second infiltration of the Castel Sant'Angelo, the next time that Ezio visits Leonardo, he will ask if Ezio is the one behind Rodrigo Borgia's demise and Ezio denies it. Then, this comes up:
Leonardo: This world gets stranger every day. I shall have to focus on my painting. I'm working on the small portrait of a woman. I am growing rather fond of it.
Ezio: Do not let a beautiful girl distract you from constructing my designs.
Leonardo: (wrapping his arm around Ezio) Have no worries. Women provide little distraction.
Ezio: Wait, I don't get it.
(Ezio gives him a weird look. Leonardo slowly removes his arm from Ezio and tucks it in his lap.)
Worse yet, that in and of itself is his attempt to woo her.
Ezio: (confused) What?
Cristina: Why are you just standing there?
Cristina's expression as she watched Ezio trip over his words during his attempted wooing is equally hilarious. Also, following Cristina's rejection, Ezio hilariously honest reply:
Ezio: Aspetta! (Wait!) I wasn't ready. I was planning on being really charming. And funny! Can I just have a second chance?
The memory "A Wolf in Sheep's Clothing" (the Lair of Romulus in the Basilica di San Pietro) is full of these.
While Ezio is standing in the middle of a group of Cardinals — the only guy wearing white (or usually non-red) in a crowd of red robes — this conversation is overheard:
Cardinal #1: His name is Ezio Auditore.
Cardinal #2: Who?
Cardinal #1: The Assassin. Killed the Banker. Some say he even walks the halls of il Vaticano with no one able to stop him.
Cardinal #2: There is hope after all! Perhaps Cesare's rule will come to an end, and the Church will be restored to all its former glory.
Ezio is chasing a corrupt Cardinal across a church rooftop. The Cardinal flees through a window as Ezio finally catches up, and promptly slams it shut in Ezio's face.
Cardinal: Sanctuary denied!
If you kill The Cardinal by throwing him off the edge:
Ezio: (catches the Cardinal by his necklace, halting his fall)
Cardinal: You saved me! Praise God!
Ezio: (yanks the key off of his neck, letting him fall to his death) Requiescat in Pace.
The very idea that a Cardinal in full garb (as opposed to the half-naked Juan Borgia) has free running skills that rival Ezio's is hilarious in and of itself.
Anyone with a rudimentary knowledge of either French, German, or both likely found one of the Doctors' parting words snicker-worthy.
Doctor:Auf Wiedersehen! ...That's French.
Which doubles as a Brick Joke, albeit a subtle one - near the beginning of the game, the technician operating the Animus briefly mentions that the translating software they're using has a tendency to glitch when trying to deal with German or French.
When hunting down the Baron de Valois to kill him, if atop the rooftop overlooking him and the captured Pantisilea, it's possible to cause the Baron de Valois to face a particular direction (by killing the arquebusier and throwing the body down), then quietly killing the other arquebusiers that come to investigate. When you throw money down at him afterward, the Baron will react like a guard in performing the "waving you along" motion even though he's not facing anyone — allowing you to (if done properly) jump down, grab Pantasilea and walk her out of there unscathed. In even more inadvertent hilarity, if you let go of her or she pushes you off, she'll immediately return to the "bound and kneeling" position.
If the player has a group of mercenaries following him, they will react to NPC's like you. If you push a minstrel away, one of the mercenaries will break off from the group to chase him, purely out of spite. Now that's going the extra mile.
Ezio drops the injured Caterina Sforza down a shaft into the "usual" hay bale and seems strangely delighted at her disapproval.
Likely because Caterina had just admitted to Ezio that the only reason she slept with him the night before the Siege of Monteriggioni was to ''really'' make sure that he'd lend her some of his mercenaries in defending Forlž against the Borgia. Sure, he'd answered, "Politics, I understand. You not need explain," but...
The best part is her calling Ezio "son of a bitch" in Italian after they leave the haystack.
Pretty much anytime there's a bandit ambush after Ezio has recruited Assassins becomes one of these. The bandits yell out in glee that they're about to strike it rich and how Ezio is a dead man, and his only response is to wave his hand. A couple of seconds later, the robbers get at best a moment to scream in terror before the Assassins massacre them.
During the final sequence, if you choose to be a little more sneaky on your approach, you will hear some of the guards talk about Cesare's downfall in Rome. Toward the end of the conversation, you get this:
Guard A: [...]Great rulers rise and fall like leaves in the wind.
Shaun: His last words were "Oh bother, I seem to be turning into a god." Which, incidentally, will be my last words.
Shaun: ...were raised by the She-Wolf. Which, incidentally, is the nickname for my girlfriend in the Hastings household. Don't ask.
Shaun: If we survive this, I'm going to go fishing here. You're not invited.
Shaun: The apprentice even got to keep the Mona Lisa. Either this was true love, or Salaž had one hell of a sugar daddynote which is played up during the Da Vinci Disappearance DLC.
Shaun: "What about those other seven children?" you may ask. Find out next week, on Romance Gone Wrong.
Shaun: [discussing Mount Vesuvius] I hear property is cheap right now, maybe we should invest.
Just before Sequence 8, The Vatican Area is unlocked, which contains The final Romulus Lair. Near the entrance, there are two drunks, and when Ezio enters, the drunks notice...
Drunk #1: (After Ezio enters) Did you see that white knight?
Drunk #2: humumumumm...
Which is made even more hilarious if you're not white, such as wearing an alt costume that's not white, or if you dyed your self different colors other than white.
And when Ezio comes back out...
Drunk #1: (After Ezio comes out) There he goes again!
Drunk #2: *More drunken Mumbling*
Drunk #1 *Sigh*...
Some of the things the guards say when Ezio is doing well in a fight:
Ezio kicks ass.
Guard, in a very coy tone of voice and backing off: Nice assassino... Put it down, yes?
Everyone else is dead
Terrified Guard: You know, I always thought you were a, uh kind and, um, noble... *Flees*
Guards sometimes claim to suffer Thanatosphobia if Ezio is winning a fight.
Early in the DLC, Leonardo da Vinci's artistic apprentice Gian Giacomo Caprotti, aka Salaž, asks Ezio if he was one of the new Pope's monks, to which Ezio answered, "My church is not of God." Salaž's response: "Outside of the church of God is the realm of men. You worship there, messere?" Later on, Salaž complains about Leonardo's penny-pinching, and Ezio is aghast that Leonardo would fall for it at all:
Salaž: He is so cheap! I have to beg him for money.
Ezio: He actually gives you coins?
Finally, after Ezio reunites with Leonardo the latter asks about Salaž, to which Ezio explained that Salaž was safely in Leonardo's workshop. Leonardo expressed relief, quickly amending that to be "about my florins," but Ezio declared, "You need not lie to me. Salaž fits you. I approve."
More humorously, it could be interpreted that Ezio's finally figured out what Leonardo meant about "women providing little distraction," three years later.
The first doctor Ezio sees informs him that someone his age could not recover normally from his wounds. Ezio leaves politely enough, but when he gets out of earshot...
Ezio: "Someone my age." Imbecille. (Jerk.) (as he climbs the tower) Ow, my back.
If you visit Leonardo after completing sequence 6 he starts talking about how general De Valois was found dead inside his own camp, to which Ezio responds with this gem "Could it have been an assassin?" Leonardo goes on (oblivious) about there being some theories that confirm it, and Ezio ends it with "You shouldn't listen to gossip Leonardo, it might get you in trouble."
One of the captains in charge of a Borgia-controlled guard tower can be found giving advice to his men from horseback, explaining to them that they may be under attack from assassins and gives them somewhat helpful hints about how to fight them. One of those tips is that they rarely go for a full-out frontal attack. Sending in yourself and all of your recruited Assassins at that moment certainly qualifies. Especially if one of the signaled Assassins rides in on a horse of their own from out of nowhere to kill him.
Ezio's taunts are pretty hilarious, but this one definitely stands out:
Which one goes first, the head, or the cunt?!
Charged attacks with Heavy Weapons. Headshotting a guard on a tower above you with a thrown Scottish claymore is both this and a Crowning Moment Of Awesome.
Overheard from a townsperson after a successful battle with guards: "This is our just reward - entrusting our security to incompetent guards!"
Ezio can fight an entire guard patrol using only kicks. That's right, you can kick an entire guard patrol to the ground.
The things the heralds announce are also prime comedy gold.
Herald: A miraculous cure for gout has been discovered! It involves the imbibing of vast quantities of beer!