Interleukin-18 (IL-18) is a pleiotropic pro-inflammatory cytokine belonging to the IL-1 superfamily. IL-18 plays an important role in host innate and adaptive immune defense but its aberrant activities are also associated with inflammatory diseases such as rheumatoid arthritis and Crohn's disease. IL-18 activity is modulated in vivo by its naturally occurring antagonist, IL-18 Binding Protein (IL-18BP). Recent crystal structures of human IL-18 (hIL-18) in complex with its antagonists or cognate receptor(s) have revealed a conserved binding interface on hIL-18. Through virtual screening of the National Cancer Institute Diversity Set II and in vitro competitive ELISA we have identified three compounds (NSC201631, NSC80734, and NSC61610) that disrupt hIL-18 binding to the ectromelia virus IL-18BP. Through cell-based bioassay, we show that NSC80734 inhibits IL-18-induced production of IFN-γ in a dose-dependent manner with an EC50 of ~250 nM. Our results and methodology presented here demonstrate the feasibility of developing small molecule inhibitors that specifically target the rather large interface of IL-18 that is involved in extensive protein-protein interactions with both IL-18BP and its cognate receptor(s). Our data therefore provide the basis for an approach by which small molecules can be identified that modulate IL-18 activity.