This study investigates the prevalence and antibiotic resistance profiles of bacteria in human semen, highlighting the risk of antimicrobial resistance (AMR) associated with the overuse of antibiotics. One hundred semen samples were collected from participants at the urology department of El-Hussien Hospital. The samples were screened for bacterial contamination, and isolated strains were tested for sensitivity against commonly used antibiotics. The Gram-positive bacteria were identified Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis along with Gram-negative bacteria, including Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Neisseria gonorrhoeae. Notably, P. aeruginosa exhibited the highest antibiotic resistance, confirmed by 16S rRNA sequencing (deposited under GeneBank accession number LC455963). Resistance genes blaTEM and blaCTX, known to contribute to extended-spectrum beta-lactamase (ESBL) production, were detected, underscoring the strain's robust resistance mechanisms. The findings underscore the urgent need for targeted surveillance systems to monitor infection sources and manage antibiotic use in healthcare settings to prevent further escalation of AMR.