Mucosa-associated invariant T (MAIT) cells are a subset of unconventional T cells displaying innate-like characteristics, with rapid secretion of pro-inflammatory cytokines and killing of bacterially infected cells. MAIT cells are abundant in the blood of humans and act as guardians of both mucosal and non-mucosal barriers. Major questions remain regarding the contribution of human MAIT cells in the immune response to invading pathogens such as Staphylococcus aureus, and role of potential bacterial immune evasion mechanisms. Here, we studied this MAIT cell-pathogen interplay using peripheral blood from healthy individuals, palatine tonsils and human organ donor matched blood, mucosal barrier, and lymphoid tissues. We also characterized the interplay between the MAIT cell response to S. aureus and the immune-evasive function of virulence factors, including the HlgAB leukocidin toxin. Peripheral blood MAIT cells respond to S. aureus leading to robust production of cytokines and granzyme B. MAIT cell activation by S. aureus is dependent both on IL-12 and IL-18, and activation via TCR triggering in response to MR1-presented antigen. MAIT cells are partially resistant to HlgAB leukocidin toxicity as compared to CD14+ monocytes. This coincides with higher expression level of CCR2, CXCR1 and CXCR2 on monocytes. Interestingly, activation of MAIT cells via TCR triggering or IL-12 and IL-18 reduces MAIT cells susceptibility to HlgAB toxicity. Additionally, MAIT cells display phenotypic and functional diversity according to their tissue localization and stimulation environment, leading to a differential susceptibility to S. aureus virulence factors.