Total 44 French mini straws (0.25ml) of frozen semen of Murrah buffalo bulls were collected from one of the frozen semen banks for evaluation of microbial load using the standard plate count (SPC) method using nutrient agar plate. These plates were incubated at 37oC for 72 hrs and examined for growth. The average colony count was calculated and bacteria were also identified as Gram positive and Gram negative. A total of 9 biochemical tests were performed to characterize the isolates. Antibiotic sensitivity test was also performed to test the sensitivity against Ampicillin, Erythromycin, Gentamycin and Spectinomycin. The results indicate that nine samples out of 44 (20.45%) were found positive for various bacterial isolates. Most frozen semen doses except two bull Nos. MR-1017 and MBF-0338 were acceptable to use for Artificial Insemination as per the Bureau of Indian Standard (BIS). None of sample was found with fungal infection. Both the Gram positive and Gram negative bacteria were found in these samples. Results of biochemical properties of bacterial isolates are summarized. Each isolates are varying in their biochemical profile, but all the isolates exhibited negative urea hydrolysis test. The results of antibiotic sensitivity pattern in bacterial isolates were also summarized. All bacterial isolates exhibited variable pattern against Ampicillin, Erythromycin, Gentamycin and Spectinomycin. However, Bacterial isolate from bull number MBF-0338 showed inhibition to all antibiotics. Among all these antibiotics all bacterial isolates are resistance against Ampicillin. The concentrations of antibiotics were 10�g, 10�g, 10�g and 100 �g respectively. The article describes detailed investigation of microbial load in frozen semen of Murrah buffalo bulls. 1344830870.php