Article history Dairy farming is an important social and economic activity in the Midwest region of Santa Catarina State, Brazil. However, despite technological advances in the productive chain, mastitis is still considered the main disease of dairy herds, due its high economic impact. Bacterial pathogens are the main cause of this disease and failures in the treatment of clinical cases are attributed in part to the antimicrobial resistance of these pathogens. As an alternative, in vitro antimicrobial susceptibility tests of strains isolated from bovine mastitis cases have been carried out; the information generated contributes to strategic treatment measures, and also to the control of microorganism resistance. Therefore, the aims of the present study were to evaluate the etiology and in vitro antimicrobial susceptibility profiles of bacterial strains isolated from cases of bovine mastitis in dairy herds from the Midwest region of Santa Catarina State, Brazil. For this purpose, 345 milk samples were collected from 70 herds located in 16 different municipalities. The main pathogens isolated were: staphylococci (55.9%), streptococci (34.8%), Corynebacterium spp. (7.8%) and Escherichia coli (1.4%). The resistance rates of staphylococcus strains to ampicillin and to gentamicin were lower (24.9%) and higher (33%), respectively, than those obtained in other Brazilian studies. The resistance profiles of streptococcus strains were similar to those described in the literature, while Corynebacterium spp., showed high resistance rates to tetracycline (85.2%). Moreover, a considerable prevalence of staphylococcus (27.5%) and streptococcus (11.7%) multiresistant strains was verified. These data reinforce the need for regional characterization of microorganisms.