"My ass is sweating. I got so much sweat dripping down my ass right now. I'm so nervous.
"This is how black people die in horror movies."
But it was a tarantula named Big Red that left Hart and Fallon crying with laughter.
With the spider sitting on Fallon's hand Irwin revealed it was only human breath that would spook it.
Hart began leaning in and yelling "Jimmy" in an attempt to spook it, as Fallon pleaded with Irwin to take it.
"That was the scariest thing I've ever done," Fallon said as Hart hunched over in fits of laughter.
The segment ended with a falcon which flew across from the audience to the arms of a terrified Fallon and Hart.
On Instagram Hart described it as "the funniest thing that I have ever been a part of".
Irwin, who appears regularly on the show, was promoting their new reality series Crikey! It's the Irwins on the Animal Planet channel.
Watch the hilarious segment below: