Staphylococcus aureus is one of the main bacteria that affect human health. Its reduced susceptibility to beta-lactam antibiotics has driven the clinical use of macrolides and lincosamides. However, the presence of macrolide-lincosamide-streptogramin B (MLSB)-resistant S. aureus strains is increasingly common. Wastewater treatment plants (WWTPs) are the main anthropogenic source of resistance determinants. However, few studies have assessed the importance of this environment on the dissemination of MLSB-resistant S. aureus strains. Thus, we aimed to evaluate the impact of a domestic WWTP on the resistance to MLSB and penicillin in S. aureus in southeast Brazil. Of the 35 isolates tested, 40.6% were resistant to penicillin. Resistance to erythromycin (8.6%) and quinolones (2.8%) was less common. Despite the low rate of resistance to clindamycin (2.8%), many isolates showed reduced susceptibility to this antibiotic (57.1%). Regarding the resistance phenotypes of staphylococci isolates, inducible MLSB resistance (D-test positive) was found in two isolates. In addition, 27 S. aureus isolates showed the ability to produce penicillinase. In this article, we report for the first time the importance of WWTPs in the dissemination of MSLB resistance among S. aureus from southeast Brazil.