In mountainous regions, diverse ecosystems provide a habitat for numerous species of organisms. In this study, we focused on ixodid ticks and their presence in the Western Carpathians, Poland. Our objectives were to investigate the impact of environmental factors on tick occurrence and activity, the prevalence of vectored pathogens, and tick hosts, and their role as reservoir organisms for tick-borne pathogens (TBPs). To this end, we collected ticks from the vegetation and from animals (Apodemus agrarius, A. flavicollis, Capreolus capreolus, Microtus spp., Myodes glareolus, Ovis aries). In addition, we collected blood samples from rodents. The collected material underwent molecular analysis, utilizing the high-throughput microfluidic real-time PCR technique, to detect the presence of TBPs. Our findings confirmed the occurrence of only two species of ixodid ticks in the study area: the dominant Ixodes ricinus, and Dermacentor reticulatus with very limited abundance. Temperature significantly influenced tick activity, and the number of I. ricinus nymphs varied with altitude. We also observed a circadian pattern of questing activity in I. ricinus ticks. The main hosts for juvenile tick stages were M. glareolus and A. agrarius, while adult stages were frequently found on C. capreolus. I. ricinus ticks collected from the vegetation were often infected with Rickettsia helvetica (up to 35.71%), Borrelia afzelii (up to 28.57%), and Ehrlichia spp. (up to 9.52%). In contrast, juvenile stages frequently carried Bartonella spp. (up to 10.00%), Mycoplasma spp. (up to 16.67%) and R. helvetica (up to 16.67%). Moreover, we detected genetic material of Mycoplasma spp. (up to 100.00%), Ehrlichia spp. (up to 35.71%), Bartonella spp. (up to 25.00%), and Borrelia spp. (up to 6.25%) in rodent blood samples. The obtained results indicate A. agrarius and M. glareolus as reservoir animals for TBPs in the studied region. Keywords: Ixodes ricinus; Dermacentor reticulatus; ticks; tick-borne pathogens