This study aims to evaluate the safety of Bifidobacterium breve BS2-PB3 as potential probiotics based on the FAO-WHO criteria. Genotypic and phenotypic approaches were employed to assess its safety profile. The presence of virulence factors, mobile elements, and antibiotic resistance genes were determined through bioinformatic analysis. Antibiotic susceptibility was evaluated using the Kirby-Bauer disk-diffusion test, in which the minimum inhibitory concentration for selected antibiotics was tested via the epsilometer test (Etest). Findings revealed resistance to mupirocin (MIC >1024 μg/mL), sulphomethoxazole (MIC >1024 μg/mL), and oxacillin (MIC >3 μg/mL) in B. breve BS2-PB3. Discrepancies between in silico predictions and wet lab experiments were observed for rifamycin and kanamycin resistance genes, the unexpressed rifamycin and kanamycin antibiotic resistance genes is caused by the lack of mutation that confer antibiotic resistance. No virulence factors were detected, and four insertion sequences were identified within the genome, located away from antibiotic resistance genes. Future studies should explore additional safety aspects such as behavior under low pH conditions and mucus adherence. For commercial purposes, the removal of identified antibiotic resistance genes using gene editing tools is recommended to enhance safety and address concerns associated with antibiotic resistance.