Two studies were conducted to evaluate the presence of tick-borne bacterial agents in Amblyomma americanum, lone star tick (LST), and various wildlife in Mississippi. Adult LSTs had DNA evidence of Ehrlichia chaffeensis (3.7%), E. ewingii (6.3%), and Borrelia lonestari (2.6%), while both larval (24.3%) and adult (43.5%) LSTs were positive for a Rickettsia spp. by polymerase chain reaction (PCR). White-tailed deer (WTD) were the only wildlife PCR positive for E. chaffeensis (18.8%), B. lonestari (3.1%), and Anaplasma phagocytophilum (3.1%). In addition, WTD had the highest seroprevalence to B. lonestari (19.3%) and E. chaffeensis (43.9%) antigens while raccoons had the highest seroprevalence to spotted fever group rickettsiae (R. parkeri antigen) (73.7%). These studies demonstrate evidence of potentially zoonotic tick-borne agents in LSTs and wildlife in Mississippi underscoring the importance of monitoring these agents for human and animal health.