Background: Bourbon virus (BRBV) is an emerging pathogen that can cause severe and fatal disease in humans. BRBV is vectored by Amblyomma americanum (lone star ticks), which are widely distributed across the central, southern, and eastern United States. Wildlife species are potentially important for the maintenance and transmission of BRBV, but little is known about which species are involved, and what other factors play a role in their exposure to BRBV. Methods: To assess the exposure risk to BRBV among wildlife in the St. Louis, Missouri, area, we collected sera from 98 individuals, representing 6 different mammalian species from two locations in St. Louis County: Tyson Research Center (TRC) and WildCare Park (WCP) from fall 2021 to spring 2023. The sera were used in a BRBV neutralization assay to detect neutralizing antibodies and RT-qPCR for viral RNA analysis. We also sampled and compared the abundance of A. americanum ticks at the two locations and modeled which factors influenced BRBV seropositivity across species. Results: In TRC, we observed a high rate of seropositivity in raccoons (Procyon lotor, 23/25), and white-tailed deer (Odocoileus virginianus, 18/27), but a low rate in opossums (Didelphis virginiana, 1/18). Neutralizing antibodies were also detected in sampled TRC bobcats (Lynx rufus, 4/4), coyotes (Canis latrans, 3/3), and a red fox (Vulpes vulpes, 1/1). The virological analysis did not detect BRBV RNA in any serum samples. In contrast to TRC, all sera screened from WCP were negative for BRBV-specific neutralizing antibodies, and significantly fewer ticks were collected at WCP (31) compared with TRC (2316). Conclusions: Collectively, these findings suggest that BRBV circulates in multiple wildlife species in the St. Louis area and that tick density and host community composition may be important factors in BRBV ecology.