Arthropod-borne viruses (arboviruses) are globally widespread, and their transmission cycles typically involve numerous vertebrate species. Serologic testing of animal hosts can provide a routine surveillance approach to monitoring animal disease systems, can provide a surveillance alternative to arthropod testing and human case reports, and may augment knowledge of epizootiology. Wild and captive ruminants represent good candidate sentinels to track geographic distribution and prevalence of select arboviruses. They often are geographically widespread and abundant, inhabit areas shared by humans and domestic animals, and are readily fed on by various hematophagous arthropod vectors. Ontario, Canada, is home to high densities of coexisting humans, livestock, and wild cervids, as well as growing numbers of arthropod vectors because of the effects of climate change. We collected blood samples from 349 livestock (cattle/sheep) and 217 cervids (wild/farmed/zoo) in Ontario (2016-2019) to assess for antibodies to zoonotic and agriculturally important arboviruses. Livestock sera were tested for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). Sera from cervids were tested for antibodies to BTV, EHDV, West Nile virus (WNV), eastern equine encephalitis virus (EEEV), Powassan virus (POWV), and heartland virus (HRTV). Fifteen (9.0%) cattle were seropositive for EHDV-serotype 2. Nine (4.2%) cervids were seropositive for arboviruses; three confirmed as WNV, three as EEEV, and one as POWV. All animals were seronegative for BTV and HRTV. These results reveal low seroprevalence of important agricultural, wildlife, and zoonotic pathogens and underline the need for continued surveillance in this and other regions in the face of changing environmental conditions.