Lactic acid-producing bacteria (LAB) are being widely studied due to their probiotic potential. The aim of the present study was to determine and identify the presence of LAB from canine vaginal samples, as well as to evaluate their probiotic in vitro potential. Ninety-four bitches were included in the study. Vaginal samples were obtained by means of a sterile swab and streaked on Man Rogosa Sharpe agar plates. A total of 100 LAB strains were obtained and submitted to Gram stains and basic biochemical tests, which included catalase, oxidase and haemolysis tests. Thirteen strains belonging to the genera Lactobacillus (n = 10), Lactococcus (n = 2) and Pediococcus (n = 1) were selected as potential probiotics and further subjected to evaluation of resistance to gastrointestinal conditions (pH, lysozyme, bile salts and hydrogen peroxide) and safety and efficacy in vitro (resistance to antibiotics and antimicrobial capacity). Only three strains, one Lactobacillus lactis and two Lactobacillus plantarum, accomplished the requirements for being considered as potential in vitro probiotics.