In recent years, significant changes have been observed in the distribution and abundance of local Dermacentor reticulatus populations. However, changes in D. reticulatus dynamics have not been studied in southeastern Poland. Our objective was to enhance our understanding of the environmental factors influencing the occurrence and density of D. reticulatus in this area. Additionally, we sought to investigate the genetic diversity of the tick population and the prevalence of tick-borne pathogens (TBPs). To this end, we established 45 study sites in the Subcarpathian province. Ticks were collected during their peak activity in both spring and autumn. A subset of randomly selected specimens underwent molecular analysis for TBPs screening, using high-throughput microfluidic real-time PCR. Positive amplicons were then sequenced, and phylogenetic analyses were conducted. Our findings confirmed the presence of D. reticulatus ticks in 24 surveyed sites, primarily concentrated in the northern and eastern parts of the region. The mean density of D. reticulatus ticks in their compact range was 5.8 ± 6.4 specimens/100 m2. Notably, air temperature and altitude emerged as significant factors influencing the species’ activity. We also identified a high prevalence of Rickettsia raoultii infections in adult D. reticulatus, reaching up to 84.21%. Additionally, 9.52% of ticks were found to be infected with R. helvetica and 4.76% with Anaplasma phagocytophilum. Furthermore, our genetic analyses confirmed the identity of D. reticulatus in the Subcarpathian region, aligning with haplotypes found in other regions of Poland, Czechia, Croatia, and Portugal. In conclusion, our study suggests that the surveyed region represents the current boundary of the compact range of D. reticulatus in Poland in which this tick species exhibits low genetic diversity and a narrow spectrum of detected TBPs.