Abstract: Aims:In this study, we sought to identify and characterize a collection of 101 lactobacilli strains isolated from natural whey starters used in Uruguayan artisan cheese production, based on their capacity to produce gamma-aminobutyric acid (GABA) and their probiotic potential. Methods and Results:The probiotic potential was assessed using low pH and bile salt resistance assays; bacterial adhesion to intestinal mucus was also evaluated. Selected strains were then identified by 16S sequencing, and their GABA-producing potential was confirmed and quantified using a UHPLC-MS system. Twenty-five strains were identified and characterized as GABA-producing lactobacilli belonging to the phylogenetical groups Lactiplantibacillus (n =19) and Lacticaseibacillus (n =6). Fifteen strains of the Lactiplantibacillus group showed a significantly higher GABA production than the rest. They showed the predicted ability to survive the passage through the gastrointestinal tract, according to the in vitro assays. Conclusions:A set of promising candidate strains was identified as potential probiotics with action on the gut-brain axis. Further studies are needed to assess their possible effects on behaviour using in vivo assay. Significance and Impact of the Study:This study shows the potential of strains isolated from local natural whey starters as probiotics and for biotechnological use in functional GABA-enriched foods formulation. © 2022 Society for Applied Microbiology.