A cross-sectional study was conducted from November 2014 to April 2015 to determine the prevalence of bovine mastitis, isolation of mastitis causing bacteria's and characterize antimicrobial susceptibility pattern at Holeta Agricultural Research Centre (HARC) Dairy Farms. Purposive sampling technique was employed and all lactating cows were involved in the study. Among 186 lactating cows consists of 92 Jersey as well as 94 cross breed (Boran X Holistein Fresian) were included in the study. Among selected cows, 131 (70.43%) cows were identified as bovine mastitis positive and out of which 10 (5.37%) had clinical mastitis and 121 (65.05%) had subclinical mastitis. The different types of bacterial species isolated in present study includes S. aureus (30.93%), staphylococcus other than S. aureus (14.43%), Str. agalactiae (5.15%), Str. Dysgalactiae (5.15%), Str. Uberis (12.37%), C. bovis (13.40%), E. coli (6.18%), P. aerogenosa (10.3%) and K. pneumonie (2.05%). Antimicrobial susceptibility testing revealed that 94% and 78.6% of total isolates were susceptible to gentamycin and trimethoprim-sulfamethoxazole, respectively which was followed by erythromycin and tetracycline with susceptibility rate of 73.8% and 69%, respectively. However, penicillin had shown susceptibility of 55.9% which was the least effective among the drugs used. In conclusion, different types of mastitis causing pathogens with variable rate of susceptibility to antimicrobials were able to identify. Therefore, antimicrobial therapy should be used after isolation of the suspected pathogen to avoid emergence of drug resistance.