In North Carolina, reported human cases of tick-borne illness, specifically Rocky Mountain spotted fever, have escalated over the past decade. To determine the relative abundance of vectors and to estimate the risk of acquiring a tick-borne illness in peri-residential landscapes, ticks were collected in Chatham County, a typical Piedmont county and, samples of the ticks were tested for infection with selected bacterial pathogens using real-time quantitative polymerase chain reaction assays. Ticks (n = 3746) were collected by flagging vegetation at 26 sites from April to July 2006. The predominant questing tick was Amblyomma americanum (98.5%) with significantly fewer Dermacentor variabilis (1.0%) and Ixodes scapularis (0.5%) collected. Spotted fever group (SFG) rickettsiae were detected in 68.2% of 1590 A. americanum with 56.4% of the molecular isolates identified as Rickettsia amblyommii, an informally named member of the SFG rickettsiae. Comparatively, smaller numbers of A. americanum contained Ehrlichia chaffeensis (1.8%) and Borrelia lonestari (0.4%). Of 15 I. scapularis nymphs tested, 6 (40%) were positive for Borrelia burgdorferi sensu lato. Seven (19.4%) of 36 adult D. variabilis tested positive for Rickettsia montanensis, 4 (11.1%) were positive for R. amblyommii, and 5 (13.9%) were infected with unidentified species of SFG rickettsiae. The tick population in Chatham County contains a diverse array of microbes, some of which are known or potential pathogens. Highest attack rates would be expected from A. americanum ticks, and highest potential risk of infection with a tick-transmitted agent would be to rickettsial organisms, particularly R. amblyommii. Accordingly, longitudinal eco-epidemiology investigations are needed to determine the public health importance of A. americanum infected with rickettsial organisms.