ABSTRACT. de Oliveira U.V., Galvão G. da S., Ribeiro A.R. da P., Andreoli J.L. & Munhoz A.D. [Efficacy in vitro of gentamicin on bacteria isolated from cows with subclinical mastite in microrregion Ilhéus-Itabuna, Bahia]. Eficácia in vitro da gentamicina sobre bactérias isoladas de vacas com mastite subclínica na microrregião Ilhéus-Itabuna, Bahia. Revista Brasileira de Medicina Veterinária, 34(3):213-218, 2012. Laboratório de Análises Clínicas Veterinárias, Departamento de Ciências Agrá- rias e Ambientais, Universidade Estadual de Santa Cruz, Rodovia Ilhéus-Itabuna, Km 16, Salobrinho, Ilhéus, BA 45662000, Brasil. Email: email@example.com The aimed this study was evaluated in vitro the sensivity to anbiotics in bacteria isolated from milk of cows diagnosed with subclinical mastitis, from 10 dairy farms, with acess available, located in the microrregion Ilhéus-Itabuna in Bahia. Twenty percent of lactating cows of each property was selected, and the selection criterion utilized was the presence of the cow in the parlor at the time of visit. The test for identification of subclinical mastitis was the California Mastitis testis (CMT), in roofs where a positive reaction was detected, proceeded to the sample, with approximately 10 mL of milk in sterile test tube with screw cap, identified and kept in refrigeration until it is processed and later made the isolation and identification of bacteria. The antibiogram was performed by disk diffusion method, and the antibiotics tested with the following concentrations per disk: ampicillin (10μg), clindamycin (2μg), chloranphenicol (30μg), gentamicin (10μg), oxacillin (1μg), penicilin (10μg), tetracycline (30μg) e vancomycin (30μg). The results was analyzed using descriptive analysis to calculate absolute and relative frequencies for the different parameters studied. Of the 187 cows selected and submitted to CMT, 74 was positive, totaling 110 teats reagents. Being identified 45 colonies of Staphylococcus aureus, 45 de Corynebacterium sp., 37 de S. coagulase negative, 16 de Bacillus sp., 16 de Escherichia coli e 2 de Klebsiella sp. Among all tested antibiotics gentamicin was the one with the lowest percentage of resistance in all bacteria isolated in this study, proved to be an alternative to producers in the region of study for the control of bovine mastitis.