"If you remain pure and stay with your faith, with what you believe in -- in my case, monsters -- you can do anything," Del Toro told the festival, the world's oldest, as he dedicated the award to young Latin American directors.
The quirky, other-worldly tale set in the Cold War era sees a mute cleaner (Britain's Sally Hawkins) in a high-security government laboratory stumble across a classified experiment that leads to an unlikely -- and rather slimy -- love affair.
Del Toro, the director behind such Gothic horrors as "The Devil's Backbone" (2001) and "Pan's Labyrinth" (2006), had described the flick as "an antidote to cynicism" and enchanted reviewers hailed it as his greatest work yet.
"I'm 52 years old, I weigh over 110 kilograms and I've done more than 10 movies," he said as he held up his Lion. Even so, he said, this had not stopped him from "doing something different".
"As a Mexican, I dedicate this award to all those Mexicans and Latin American directors dreaming of doing something as a parable, who are told it can't be done. It can be done."
"I believe in life, in love and in cinema," he added at the close of a ceremony heavy with emotion.
Tears and cocktails
French director Xavier Legrand snapped up two prizes, breaking down and sobbing openly as he was awarded the Silver Lion for best director hot on the tail of picking up the Lion of the Future for best debut film.
He said his divorce tale "Custody", in which a child is held hostage to the escalating conflict between his parents, had been "a tale that urgently needed to be told" and a fierce denunciation of violence against women.
Best screenplay went to British-Irish Martin McDonagh for "Three Billboards outside Ebbing, Missouri", a dark comedy about a grieving mother who takes on the town's police chief when he fails to solve her daughter's murder.
"We've had a beautiful time (here in Venice), some beautiful pasta, some beautiful Negronis (cocktails), but this is the best part," said McDonagh, who directed "In Bruges" (2008) and "Seven Psychopaths" (2012).
Britain's Charlotte Rampling won best actress for her role in Andrea Pallaoro's "Hannah", and said it was a "huge honour" because Italy, where she did her first film in 1982, "is the source of absolutely all of my inspiration".
Best actor went to Kamel El Basha in Ziad Doueiri's Lebanese film "The Insult", a tale of a spat which escalates and lands a Lebanese Christian and Palestinian refugee in court. And Samuel Maoz's "Foxtrot", a surreal, off-balance family tragedy in three acts described by the Israeli director as "a dance of a man with his fate", snapped up the Grand Jury prize.
For the first time, the festival included virtual reality films in the competition. The best VR award went to American Eugene YK Chung, with "Arden's Wake Expanded".
By AFP's Catherine Marciano with Ella Ide in Rome