A total 20 French mini straws (0.25ml) of frozen semen were randomly collected from one of frozen semen bank for evaluation of microbial load using the standard plate count (SPC) method using nutrient agar plate. These plates were incubated at 37oC for 24 and 48 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 10 biochemical tests were performed to characterize the isolates. Antibiotic sensitivity test was also performed to test the sensitivity against Ampicillin, Erythromycin, Gentamycin, Spectinomycin and Tetracycline. The results indicate that 5 samples out of 20 were found positive for various bacterial isolates and fungi. Both the Gram positive and Gram negative bacteria were found in these samples. One isolate from sample No. CB-594 (white colony) was found positive for all 9 biochemical tests. All the bacterial isolates exhibited variable pattern against 5 antibiotics used in the study. The article describes detailed investigation of microbial load in frozen semen of cattle bulls. 1308233242.php