Pathogens such as herpesviruses, Mycoplasma spp., and frog virus 3-like ranavirus have contributed to morbidity and mortality in many species of free-living and zoo-maintained chelonians. However, their prevalence is understudied in Blanding's turtles (Emydoidea blandingii) across North America. To assess the presence of these pathogens, Blanding's turtles were sampled in Lake County, Illinois, in 2017 (N = 213) and 2018 (N = 160). DNA from cloacal-oral swabs was assayed for four ranaviruses, three Mycoplasma spp., two Salmonella spp., Emydoidea herpesvirus 1 (EBHV1), and tortoise intranuclear coccidiosis (TINC) using a multiplex quantitative polymerase chain reaction (qPCR). Pathogens were most frequently detected in adult turtles (n = 25) and rarely in subadults (n = 2) or juveniles (n = 1). EBHV1 was detected in 22 individuals with no clinical signs of illness, most (n = 20) occurring in the month of May (P < 0.0001). EBHV1 cases at one study site significantly clustered within the same 0.64-km area from 17 to 22 May 2017 (P < 0.0001) and 14 to 15 May 2018 (P = 0.0006). Individuals were rarely positive for Salmonella typhimurium (n = 6). A novel Mycoplasma sp. sharing high homology with other emydid Mycoplasma spp. was detected in one turtle with nasal discharge. Neither TINC nor any ranaviruses were detected. Continued monitoring of this population and habitat may facilitate identification of risk factors for pathogen occurrence and clarify the impact of infectious diseases on Blanding's turtle conservation outcomes.