Records of in vitro susceptibility tests performed between 1992 and 2011 were retrospectively reviewed in order to evaluate the dynamic profiles of possible changes in antimicrobial resistance of Staphylococcus spp. isolated from milk samples of cows with mastitis during two decades. The results of 2,430 isolates tested by disk diffusion technique for susceptibility to oxacillin, penicillin, ampicillin, cephalexin, norfloxacin, tetracycline, sulfazotrim, gentamicin, and neomycin were analysed. Comparisons were performed between the percentages of resistance to antimicrobials and their classes and also between the decades studied. Additionally, the possible tendency or changes in the behaviour of these pathogens against the major drugs used in the last two decades were evaluated using regression analysis. The highest rates of resistance (P<0.0001) were observed for the beta-lactams (34.3%), with exception of cephalexin (6.9%), and for the tetracyclines (28%). Similar resistance rates (7.6% to 15.7%) were observed among the other drugs. Regression analysis showed a reduction in resistance to penicillin and ampicillin throughout the period, whilst for oxacillin and neomycin a decrease in the resistance was observed during the first decade, followed by an increase. A trend towards decreased resistance was found for sulfazotrim, whereas for the other antimicrobials no decrease was observed. The results indicated no trend towards increased resistance for most antimicrobials tested. Nevertheless, it is necessary to monitor the resistance patterns of these pathogens in order to save these drugs as a therapeutic reserve