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In vitro fertilization (IVF) is the most advanced technique of assisted reproductive where the sperm reach the eggs for fertilization in a laboratory tube. Seminal fluid often contains bacterial contaminants that decrease the quality and quantity of seminal fluids. Aim: In this study we investigated the presence of bacteria in semen before IVF procedure. Methodology: All samples were identified according to the morphological, biochemical, molecular, and antimicrobial tests. Results: Microbial species were detected in 28% of semen samples, while 72% did not show any bacterial growth. The prevalent bacterial isolates were Staphylococcus spp., Enterococcus spp., Catellicoccus spp., and Pseudomonas spp. Conclusion: The use of microbiological analysis to identify resistant bacterial isolates is considered an effective method to reduce infertility in men by changing the contaminated semen.