She and Phillip Schofield had been hosting a segment about guide ponies as an alternative to guide dogs when one of the animals suddenly relieved itself on the floor.
Schofield had been discussing the issue of ponies going to the toilet on the floor of a person's house when the messy moment happened.
Willoughby immediately fell about in fits of laughter, tears streaming down her face and causing her mascara to run.
Schofield said, stifling a laugh: "There we go, perfect timing, absolutely perfect timing.
"I'm sure a person who had impaired sight would find that extremely useful on the kitchen rug. Well done – I think you've proved my point here that there are limitations between horses and dogs."
Willoughby was unable to speak for several moments while trying to maintain her composure.
She eventually said, wiping her eyes: "That was my favourite thing I've ever seen."
The trainer who had accompanied the horse on screen said: "He just wants to make himself known on TV for the wrong reasons."
For the remainder of the segment, which saw the trainer speak of the positives of having a guide pony, Willoughby struggled to keep a straight face.
The TV presenter said: "I'm so sorry, it's just because he's done a poo on the floor and it's sent us all a bit loopy!"
A member of the TV crew had to come onto the set to clean up the mess with two large bin bags, before a demonstration of how the miniature horse could aid a person in need took place.
Viewers were left in hysterics as they took to Twitter to share their amusement over the animal's faecal faux pas.
One wrote: "I can't believe I am blessed enough to have watched that guide pony disaster live on #ThisMorning."
Another added: "Couldn't stop laughing about what the horse had just done live on TV #ThisMorning."
One viewer said they had "tears streaming" down their face, while one wrote that "Holly's reaction to the miniature ponies on @thismorning is the funniest thing ever!".
"What a classic on #ThisMorning I was wondering if one of those pony would do one, priceless," one viewer declared.
For many TV fans, the mishap brought back memories of the moment an elephant went to the toilet during Blue Peter during a 1969 episode.
One commented: "I can't stop laughing at @thismorning having their Blue Peter 'Pony Poo' moment! #PonyPoo #GuidePony #ThisMorning."
Another said it was the "funniest thing to happen on TV" since the elephant incident.