Background Staphylococcus aureus (S. aureus) is a pathogen responsible for a wide range of clinical manifestations and potentially fatal conditions. There is a paucity of information on the influence of androgens in the immune response to S. aureus infection. In this study, we evaluated the influence of the hormone 5α-dihydrotestosterone (DHT) on mouse peritoneal macrophages (MPMs) and human peripheral blood monocytes (HPBMs) induced by S. aureus. Methods An in vitro model of MPMs from BALB/c sham males, orchiectomised (OQX) males, and females was used. Cells were inoculated with 10 μL of S. aureus, phage-type 80 or sterile saline (control) for 6 h. The MPMs of OQX males and females were pre-treated with 100 μL of 10–2 M DHT for 24 h before inoculation with S. aureus. The concentration of the cytokines TNF-α, IL-1α, IL-6, IL-8, and IL-10; total nitrites (NO−2); and hydrogen peroxide (H2O2) were measured in the supernatant of MPM cultures. In addition, the toll-like receptor 2 (TLR2) and nuclear factor kappa B (NF-kB) genes that are involved in immune responses were analysed. For the in vitro model of HPBMs, nine men and nine women of childbearing age were selected and HPBMs were isolated from samples of the volunteers’ peripheral blood. In women, blood was collected during the periovulatory period. The HPBMs were inoculated with S. aureus for 6 h and the supernatant was collected for the analysis of cytokines TNF-α, IL-6, IL-12; and GM-CSF, NO−2, and H2O2. The HPBMs were then removed for the analysis of 84 genes involved in the host’s response to bacterial infections by RT-PCR array. GraphPad was used for statistical analysis with a p value < 0.05. Results Our data demonstrated that MPMs from sham males inoculated with S. aureus displayed higher concentrations of inflammatory cytokines and lower concentrations of IL-10, NO−2, and H2O2 when compared with MPMs from OQX males and females. A similar result was observed in the HPBMs of men when compared with those of women. Previous treatment with DHT in women HPBMs increased the production of pro-inflammatory cytokines and decreased the levels of IL-10, NO−2, and H2O2. The analysis of gene expression showed that DHT increased the activity of the TLR2 and NF-kB pathways in both MPMs and HPBMs. Conclusions We found that DHT acts as an inflammatory modulator in the monocyte/macrophage response induced by S. aureus and females exhibit a better immune defence response against this pathogen.