This study aimed to isolate and characterize in-vitro and under simulated colonic conditions, probiotic candidates isolated from food environment producing γ-aminobutyric acid (GABA), a major inhibitory neuromediator of the enteric nervous system with a potential role in modulating the immune system in many health disorders. Several lactic acid bacteria were isolated and detected for the presence of the gadB gene using PCR and GAD enzymatic assay. The most active strains with high and fast production kinetics were identified, characterized, and included Streptococcus thermophilus, Lactiplantibacillus plantarum, and Lactobacillus delbrueckii subsp. bulgaricus. The biological safety (i.e., sensitivity to antibiotics and the presence of virulence factors) and probiotic potential (i.e., resistance to gastrointestinal conditions and whole-genome sequencing) of identified bioactive strains was also confirmed in vitro. The growth, GABA production, and competitiveness of selected probiotic candidates (B. animalis, S. thermophilus, and L. bulgaricus) were investigated in the presence of human gut microbiota ex vivo in a model of a proximal colon mimicking physiological and microbiological conditions of the human large intestine. Supplementation with GABA-producing probiotic candidates did not affect the overall gut microbiota diversity over 48 h of treatment. However, we observed modulation of the microbiome composition, especially change of Bacteroides population, a key gut microbe associated with anti-depressive and anti-inflammatory activities. The level of microbiota-generated butyrate within 12 h of treatment was significantly increased compared to control. Results from this study demonstrated the probiotic potential of tested GABA-producing bacteria and their impact on gut microbiota structure and metabolism, suggesting their suitability for gut health-promoting application.