The 175-km ride from Imperia to Novi Ligure looked to be headed for a sprint finish before Segaert made his telling move 3 km from the line and held off the chasing pack to take the win on his Giro debut.
Fellow Belgian Toon Aerts (Lotto-Intermarche) won the dash for second ahead of one-time race leader Guillermo Thomas Silva (XDS Astana Team).
Segaert was asked when he thought about making his attack.
"I would say yesterday evening," Segaert said with a smile.
"I had it always in mind, I was really happy with how the race was going, a hard pace on the climb and then teammates of the sprinters who were left had to ride hard and this was my chance to go in the final when they were all on the limit."
Portugal's Eulalio put an extra six seconds between himself and favourite Jonas Vingegaard, with the Dane now 33 seconds off the pink jersey.
Johan Jacobs attacked from the start, and while his breakaway companions changed several times, the Swiss rider was still two minutes ahead of the peloton after 100 km as the race reached the first of the day's two climbs.
Movistar, yet to win a stage in this year's Giro, began to drive the main bunch once they hit the ascent and the leaders were reeled in as they reached the top of Colle Giovo.
Several riders were dropped on the second climb, with Movistar working hard at the front for Orluis Aular, who finished second to Jhonatan Narvaez on stage four, and teammate Enric Mas also lost out to the Ecuadorean on Wednesday.
Every second counts
Those dropped by the peloton included points classification leader Paul Magnier and Italian Jonathan Milan, and despite the sprint rivals working together to bridge the gap, they realised their race was done for the day.
Eulalio was first to surprise the bunch at the intermediate sprint, and Giulio Ciccone tried to get away with 7 km left but the Italian's attack was quickly snuffed out.
Segaert was next to go, catching Vingegaard and his Visma-Lease a Bike team off guard, and the sprinters were denied their expected battle. After all their work, Movistar's Aular had to settle for sixth on the line.
Friday's stage 13 takes the riders 189 km from Alessandria to Verbania.
