Babesiosis is an emerging tick-borne zoonotic disease in the United States caused by Babesia parasites. In 2009, the first case of babesiosis was documented in Tennessee. Environmental investigation at the reported site of tick exposure included collection of ticks and specimens from eastern cottontail rabbits (Sylvilagus floridanus) and white-tailed deer (Odocoileus virginianus) that were tested for piroplasms by molecular and serologic methods. One hundred and sixty-six Ixodes scapularis ticks and biological samples from 8 rabbits and 5 deer were collected. Ixodes scapularis were PCR positive for Babesia odocoilei (n = 7, 4%) and Theileria cervi (n = 24, 14%). Deer were seropositive for B. odocoilei and PCR positive for T. cervi. Rabbits were seropositive for B. odocoilei and Babesia sp. MO1, and 1 rabbit was PCR positive for Babesia sp. MO1. In summary, zoonotic Babesia sp. MO1 infection in rabbits is reported here for the first time in Tennessee as well as infection of deer and I. scapularis ticks with 2 other piroplasms of veterinary importance.