The aim of this study was to evaluate the probiotic potential of Lactobacillus brevis G145 isolated from traditional Khiki cheese, analyzing pH and bile resistance, physicochemical properties of the strain (hydrophobicity, auto- and co-aggregation), cholesterol removal, hydroxyl radical scavenging activity, adhesion ability to Caco-2 cells monolayer, and adhesion competition on Enterobacter aerogenes (competition, inhibition and replacement assays). Also, DNase, haemolytic activity, biogenic amine production and antibiotic susceptibility was investigated. L. brevis G145 was resistant to acidic pHs, bile salts, and simulated gastrointestinal conditions, and showed remarkable cell surface hydrophobicity (49.56%), co-aggregation (28.90%), auto-aggregation (34.10%), adhesion (9.40%), cholesterol removal (45.50%), and antioxidant (52.19%) properties. According to the results of well diffusion agar and disc diffusion agar tests, the highest and lowest inhibition zones were accounted for Staphylococcus aureus and Enterobacter aerogenes, respectively. The isolate did not show haemolytic, DNAse, and biogenic amine production properties. It was sensitive to antibiotics erythromycin, ciprofloxacin, and chloramphenicol, and semi-sensitive to imipenem, ampicillin, nalidixic acid, and nitrofurantoin. According to the findings of probiotic tests L. brevis G145 can be used as a in the food industry.