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Despite the increasingly recognized eco-epidemiological importance of ticks as vectors for numerous zoonotic pathogens in urban areas, data regarding the pathogen diversity and co-infection rates in ticks and wildlife hosts in urban and peri-urban Romania are scanty. We aimed to establish the risk of human exposure to co-infected ticks in Cluj-Napoca, a major city in Romania. DNA was isolated from 151 questing ticks:  Ixodes ricinus  ( n  = 95),  Haemaphysalis punctata  ( n  = 53),  Dermacentor reticulatus  ( n  = 2), and  Dermacentor marginatus  ( n  = 1); 222 engorged ticks:  I. ricinus  ( n  = 164),  I. hexagonus  ( n  = 36),  H. punctata  ( n  = 16),  H. concinna  ( n  = 6), and 70 tissue samples collected from wildlife hosts during 2018 in five urban, and two peri-urban sites. Using a pre-designed Fluidigm real-time PCR dynamic array, all DNA samples were individually screened for the presence of 44 vector-borne pathogens. Subsequently, conventional PCRs were performed for a selection of samples to allow validation and sequencing. In total, 15 pathogens were identified to species and 6 to genus level. In questing ticks, single infections were more common than co-infections. Seven  Borrelia  spp. were detected in questing  I. ricinus , and three in  H. punctata  ticks. An overall high prevalence 26.35% (95% CI: 19.46–34.22) and diversity of  Borrelia burgdorferi  sensu lato was seen in urban questing ticks. Other pathogens of the order Rickettsiales were present with variable prevalence. Co-infections occurred in 27.4% (95% CI: 18.72-37.48) of all infected questing ticks. In engorged ticks the overall  Bo. burgdorferi  sensu lato prevalence was 35.6% (95% CI: 29.29–42.27), with five species present. Pathogens of the order Rickettsiales were also frequently detected. We report for the first time in Romania the presence of  Rickettsia aeschlimannii  and  Rickettsia felis . Overall, from the infected engorged ticks, 69.2% showcased co-infections. In  Ixodes  spp., dual co-infections, namely  Borrelia  spp. and  Anaplasma phagocytophilum , and  Rickettsia helvetica  and  A. phagocytophilum  were the most prevalent. Given the outcome, we underline the need to establish proper tick-surveillance programs in cities and include co-infections in the management plan of tick-borne diseases in Romania.

High Diversity, Prevalence, and Co-infection Rates of Tick-Borne Pathogens in Ticks and Wildlife Hosts in an Urban Area in Romania