Abstract Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp. and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, P. membranifaciens, Saccharomyces cerevisiae and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against A. flavus and A. parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them ‒belonging to S. cerevisiae and P. kudriavzevii species‒ generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence related traits, growth at high temperatures, enzymatic activities and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.