Mastitis is the most prevalent disease of dairy cows mainly caused by bacteria. Antimicrobials are largely used in dairy farmers for prophylactic control of mastitis as dry cow therapy. The use of antimicrobials in dairy cows for treatment of mastitis has improved their health and productivity, but prophylactic usage is under scrutiny due to the risk of antimicrobial resistance (AMR). AMR is a threat to animal and human health due to rising reservoir of resistant population of pathogenic bacteria. The aim of this study was to determine sensitivity of bacterial isolates from cases of bovine mastitis to commonly used antimicrobials. In total, 174-quarter milk samples from 148 cows, 3 composite milk samples from 3 cows and 1 bedding sample were collected from 35 dairy farms in Tennessee, Kentucky, and Mississippi. We tested isolates for sensitivity with a panel of 10 antimicrobials by MIC using the Sensititer system. We used descriptive statistics and logistic regression for data analysis. Overall, we isolated 197 isolates, comprising of 34% Staphylococcus aureus, 21.3% Streptococcus uberis, 18.3% Streptococcus dysgalactiae, 17.8% Escherichia coli, 6.6% Klebsiella pneumoniae and 2% Klebsiella oxytoca. The most prevalent pathogen at cow level was S. aureus followed by Str. uberis, Str. dysgalactiae, E. coli, K. pneumoniae and K. oxytoca, whereas at the farm level Str. uberis was the most prevalent followed by Str. dysgalactiae, S. aureus, E. coli, K. pneumoniae and K. oxytoca. Cows infected with E. coli mainly had clinical mastitis, whereas those infected with S. aureus had subclinical mastitis (P < 0.05). Ceftiofur showed the highest efficacy on the most isolates followed by cephalothin, but K. pneumoniae isolates were resistant to most of the antimicrobials tested. Isolates from subclinical mastitis had higher MIC (P > 0.05) compared with isolates from clinical mastitis for some antimicrobials. The 197 isolates showed 32 different AMR patterns, which varied with farms and states. Compared with S. aureus and Str. dysgalactiae, E. coli and Klebsiella spp. had widespread resistance to pirlmycin and higher MIC for most of the drugs tested. These results suggested that confirmatory diagnosis and subsequent sensitivity testing would be a prerequisite to treat these mastitis pathogens effectively.