The escalating incidence of hospital infections due to antibiotic resistance necessitates the identification of alternative therapeutic agents such as probiotics. This study was designed to isolate and evaluate the efficacy of probiotics against Staphylococcus saprophyticus, a prevalent etiological agent of urinary tract infections (UTIs). A total of 100 S. saprophyticus strains were isolated from clinical samples and subjected to antibiotic susceptibility testing via the disc diffusion method. Concurrently, probiotic bacteria were isolated from Bulgarian cheese and shallot yogurt, and their antibacterial activity against S. saprophyticus strains was assessed. The inhibitory potential of probiotic supernatants was evaluated using microtiter plate assays, with the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determined at a 1/2 dilution. Cytotoxicity was evaluated using the MTT assay, and high‐performance liquid chromatography (HPLC) was employed to analyze the concentrations of organic acids produced by the probiotics. The results revealed that all S. saprophyticus strains were resistant to tetracycline and doxycycline but susceptible to other antibiotics. Lactobacillus rhamnosus strains M and B demonstrated notable antibacterial and antibiofilm activity against S. saprophyticus isolates. These probiotics exhibited susceptibility to most antibiotics and lacked virulence factors, suggesting their safety for therapeutic use. The organic acids produced by the probiotics were identified as lactic acid, acetic acid, and formic acid. In conclusion, L. rhamnosus strains M and B exhibit potent antimicrobial properties against S. saprophyticus, indicating their potential as therapeutic agents for UTIs. Further research is warranted to validate these findings and explore the possibility of these probiotics in clinical applications.
