The presence of harmful microbes may negatively influence semen quality. Antibiotic used in the semen extender should be evaluated in order to prevent contamination and infertility. In this study, Antibiogram evaluation of Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus epidermidis and Staphylococcus intermedius were detected from frozen semen of cattle. Different antibiotics such as amikacin, ampicillin, amoxicillin, cephalexin, erythromycin, gentamicin, neomycin, ofloxacin and sulphamethoxazole/trimethoprim were tested against the isolated bacterial species. Ofloxacin, amikacin, cephalexin and amoxicillin were highly effective against Micrococcus luteus. The organism was found equally effective to erythromycin, gentamycin and neomycin. Amikacin, ofloxacin, erythromycin, neomycin and ampcillin were the most effective against the Pseudomonas aeruginosa. However, amoxicillin, cephalexin and