A header from Kleber in the 74th minute gave Estoril a deserved victory at home on Saturday, as Vitoria's winless run stretched to three matches.
The victory extended Estoril's unbeaten run in all competitions to seven games, as Jose Couceiro's men moved up to ninth in the table with 18 points.
Estoril tested visiting goalkeeper Assis on a number of occasions at the Estadio Antonio Coimbra da Mota, taking five shots with three on target, compared to just two from the visitors.
But it was not until Kleber nodded a right-wing cross into the net from the edge of the six-yard box that Estoril moved ahead.
Vitoria have been top of the Portuguese first division on five occasions so far this season but their loss at Estoril leaves them six points behind leaders Benfica with the Lisbon-based giants still to play this weekend.
They could even head into the festive break fourth in the table if Sporting Braga beat Pacos de Ferreira on Monday.