Nystatin is an antifungal compound with potent proinflammatory properties. Herein, we demonstrate that nystatin induces interleukin (IL)-1beta, IL-8, and tumor necrosis factor alpha secretion through its activation of toll-like receptor 1 (TLR1) and TLR2. Hence, a TLR-dependent mechanism could serve as the molecular basis for the proinflammatory properties of nystatin.