Objectives: The Balkan Peninsula, acting as a crossroad between central Europe and the Middle East, presents diverse ecosystems supporting various tick species capable of transmitting TBDs. This study focuses on Serbia and North Macedonia, both endemic for TBDs, aiming to investigate human-biting ticks' prevalence, TBD prevalence, and major TBPs in blood samples. Patients and Methods: This prospective observational study was conducted in 2022 at two medical centers, involving 45 patients from Novi Sad, Serbia, and 17 patients from Skopje, North Macedonia. All participants had either a tick still attached or had had one removed within the preceding 48 h. The study consisted in clinical evaluations of patients and testing of patient samples and ticks for tick-borne pathogens using a High-Throughput pathogen detection system based on microfluidic real-time PCR. In addition, the study assessed the genetic diversity of the identified pathogens. Results: Ixodes ricinus was the most prevalent tick species, with varying infestation rates across various body parts. Tick species and feeding times differed between Novi Sad and Skopje. TBPs were prevalent, with Rickettsia spp. dominant in Skopje and a mix including Rickettsia aeschlimannii, , Rickettsia monacensis, , Anaplasma phagocytophilum, , and Borrelia afzelii in Novi Sad. Subclinical bacteremia occurred in 8.06% of cases, mostly involving Anaplasma spp. Clinical manifestations, primarily local hypersensitivity reactions, were observed in six patients. Phylogenetic analysis confirmed R. aeschlimannii and R. monacensis identity, highlighting genetic differences in gltA gene sequences. Conclusions: This study sheds light on the prevalence and diversity of TBPs in tick-infested individuals from Serbia and North Macedonia, contributing valuable insights into the epidemiology of TBDs in the Balkan region.