Determining which wildlife hosts are involved in the enzootic cycles of tick-borne diseases (TBD) enables enhanced surveillance and risk assessment of potential transmission to humans and domestic species. Currently, there is limited data to indicate which tick-borne pathogens (TBP) can infect coyotes. Additionally, limited surveillance data for white-tailed deer (WTD) in south Texas is available. The purpose of this study was to detect current infections of common TBP in coyotes and WTD in south Texas, which represents a transboundary region and common site for animal migrations across the U.S.-Mexico border. A patent pending real-time PCR assay, the TickPath layerplex test, was used to screen whole-blood samples for species from Borrelia, Rickettsia, Ehrlichia, Anaplasma, and Babesia genera. Conventional PCR and subsequent sequencing of positive samples confirmed the pathogen species. Of 122 coyote samples, 11/122 (9.0%) were positive for Babesia vogeli and 1/122 (0.8%) was positive for Borrelia turicatae. Of 245 WTD samples, 1/245 (0.4%) was positive for Anaplasma platys, 4/245 (1.6%) were positive for Ehrlichia chaffeensis, and 18/245 (7.3%) were positive for Theileria cervi. All positive samples from both species, except for one coyote, were collected from counties located in south Texas along the U.S.Mexico border. One coyote positive for B. vogeli originated from a county in northern Texas. The results from this study depicts the first known molecular detection of B. turicatae in a coyote, and demonstrates that coyotes and WTDs can potentially serve as sentinels for several zoonotic TBD as well as TBD that affect domestic animals.