Abstract Ixodes ricinus is the principal vector of numerous tick-borne pathogens (TBPs) in Europe and is typically associated with forest sites that provide favorable microclimatic conditions. However, this species may also occur in meadow ecosystems, which are generally regarded as suboptimal environments and remain insufficiently studied from an epidemiological perspective. The aim of this study was to determine and compare the prevalence of Borrelia spp. and Rickettsia spp. in adult I. ricinus occurring in urban and rural meadow sites in eastern Poland. Ticks collected between June 2023 and May 2024 were screened for Borrelia spp. and Rickettsia spp. using high-throughput microfluidic real-time PCR targeting the 23S rRNA and ITS regions, respectively. The DNA of Borrelia spp. was detected in 14.8% of ticks from the urban site and 10.7% from the rural site, whereas Rickettsia spp. were detected in 5.6% and 8.9% of specimens, respectively. No significant differences in pathogen prevalence were observed between sites. The results confirmed the presence of Borrelia spp. and Rickettsia spp. in adult I. ricinus collected in urban and rural meadow sites. Keywords: tick-borne pathogens; ticks; Ixodes ricinus; Borrelia spp.; Rickettsia spp.; suboptimal meadow sites