Urbanization alters components of natural ecosystems which can affect tick abundance and tick-borne disease prevalence. Likely due to these changes, tick-borne pathogen prevalence has increased in many U.S. urban areas. Despite the growing public health importance of tick-borne diseases, little is known about how they are influenced by urbanization in North America, especially in the central U.S. where several pathogens occur at or near their highest levels of incidence nationally. To determine whether urban development influences tick infection with bacteria and protozoa, we collected ticks at 16 parks across a gradient of urbanization intensity in Oklahoma City, Oklahoma, USA and tested them using a variety of PCR assays. Adult ticks tested positive for Rickettsia parkeri, R. amblyommatis, R. rhiphicephali, ‘Candidatus R. andeanae’, Ehrlichia chaffeensis, E. ewingii, Panola Mountain Ehrlichia, ‘Borrelia lonestari’, Theileria cervi, Babesia spp. Coco, and Cytauxzoon felis. These results indicate the presence of a high diversity of tick-borne bacteria and protozoa across an expanding urban area in the U.S. Great Plains. Although there appeared to be some risk of encountering tick-borne microorganisms across the entire urbanization gradient, E. chaffeensis, E. ewingii, and T. cerviinfected ticks and microbe diversity decreased with increasing urbanization intensity. We identified a low rate of coinfection between different microorganisms, with coinfected ticks mainly collected from sites in the least-urbanized areas. This study suggests the need for awareness of tick-borne disease risk throughout urban areas in the central U.S., and highlights a need for studies of tick host habitat use and movement in cities.