Staphylococcus aureus (S. aureus) is one of the most serious human pathogens. α-Hemolysin (Hla) secreted by S. aureus is a key toxin for various infections. We herein report that Honokiol, a natural plant polyphenol, inhibits the secretion and hemolytic activity of staphylococcal Hla with concomitant growth inhibition of S. aureus and protection of S. aureus-mediated cell injury within subinhibitory concentrations. In parallel, Honokiol attenuates the staphylococcal Hla-induced inflammatory response by inhibiting NLRP3 inflammasome activation in vitro and in vivo. Consequently, the biologically active forms of the inflammatory cytokines IL-1β and IL-18 are reduced significantly in response to Honokiol in mice infected with S. aureus. Experimentally, we confirm that Honokiol binds to monomeric Hla with a modest affinity without impairing its oligomerization. Based on molecular docking analyses in silico, we make a theoretical discovery that Honokiol is located outside of the triangular region of monomeric Hla. The binding model restricts the function of the residues related to membrane channel formation, which leads to the functional disruption of the assembled membrane channel. This research creates a new paradigm for developing therapeutic agents against staphylococcal Hla-mediated infections.