The role of wildlife in the complex balance of tick-borne diseases within ecosystems is crucial, as they serve as hosts for tick carriers and reservoirs for the pathogens carried by these ticks. This study aimed to investigate the presence of zoonotic pathogenic bacteria in wildlife, specifically in hares and long-eared hedgehogs (Hemiechinus megalofis), in the eastern region of Iran. The focus was on the detection of Borrelia spp., Coxiella burnetii, Anaplasma spp., Francisella spp., and Leptospira spp., using the Nested-PCR method. We analyzed a total of 124 blood samples, and 196 ticks collected from hares and long-eared hedgehogs were analyzed. The Nested-PCR method was employed to identify the presence of zoonotic pathogenic bacteria DNA. Our study revealed the presence of these zoonotic pathogenic bacteria in both wildlife species, indicating their potential role as hosts and reservoirs for the ticks carrying these pathogens. The specific presence and prevalence of Borrelia spp., Coxiella burnetii, Anaplasma spp., Francisella spp., and Leptospira spp. were determined through the Nested-PCR method. This study contributes to the limited knowledge about the involvement of wild animals in the transmission of tick-borne diseases. By using the Nested-PCR method, we successfully identified the presence of zoonotic pathogenic bacteria in hares and long-eared hedgehogs. This study emphasizes the need for further research to better understand the ecological process of tick-borne diseases, particularly the role of wildlife in their spread. Such knowledge is crucial for wildlife conservation efforts and the management of tick-borne diseases, ultimately benefiting both animal and human health.