The process of teat disinfection is a widely accepted component of successful mastitis control programs by reducing the number of bacteria on the teat skin and healing teat lesions. For contagious pathogens such as Staphylococcus aureus, post-milking teat disinfection remains a simple, effective and economical practice for prevention of new intramammary infections (IMIs) of lactating dairy cows. Despite the universal acceptance of teat dipping as a method of mastitis control, variations in the susceptibility and resistance profile of mastitis pathogens among antiseptics have been described. Thus, here we sought to explore the in vitro efficacy of the followings antiseptics against S. aureus isolated from IMIs: chlorhexidine (2.0%), chlorine (2.5%), quarternary ammonium (4.0%), lactic acid (2.0%) and iodine (0.6%). We used 50 S. aureus strains isolated from bovine IMIs from 50 dairy herds located at Minas Gerais, São Paulo, Paraná and Rio Grande do Sul States (Brazil). The antiseptics were evaluated at four different specific intervals (15, 30, 60 and 300 s). We found a higher activity for quarternary ammonium and chlorhexidine against S. aureus at all time-points, followed by iodine and then chlorine. Lactic acid treatment produced the worst results for all time-points and strains. Due to variations in the sensitivity and resistance profile of antiseptics against S. aureus isolated from IMIs, the effectiveness of the antiseptics against the major mastitis pathogens should be periodically evaluated in dairy farms in an attempt to reduce the rate of new IMIs in the herd.