Considering the emergence of the fact that the pathogenic bacteria may be an additional negative factor influencing male fertility and worsening sperm quality. The isolated Staph. aureus and E.coli were found resistant to the antibiotics used in treatment . This study was conducted to evaluate the using probiotic of Lactobacillus acidophilus CL1285® alternative antibiotic against Staphylococcus aureus and Escherichia coli causing urogenital tract infections in vitro. The growth inhibitory effect produced by Lb. acidophilus CL1285 on the pathogenic Staph. aureus and E.coli was tested on solid medium using agar diffusion methods. In the latter instance, the direct effect of Lb. acidophilus and bacteriocin on pathogenic isolates led to the growth inhibition of the bacterial cells after 24 h of their incubation at 37 °C. Lb.acidophilus CL1285 bacteriocin was purified by gel filtration and it increased in specific activity by antibiogram . The purified bacteriocin recorded an apparent four polypeptides with molecular weights,63,45,38,and 30 KDa. Amino acid analysis showed that the bacteriocine consists of 14 amino acids with high content of alanine, asparagine. glycine, tryptophane and glutamic acid . The results suggest that Lb. acidophilus and Cell Free Supernatants (CFS) containing bacteriocin of L. acidophillus inhibited Staph. aureus and E. coli pathogenic bacteria. So Lb. acidophillus are meant to treat infections in male urogenital tract infections; however more studies including in vivo need to be performed.