The presented study aimed at establishing the prevalence and co-infection rates of Bartonella henselae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from the central and eastern parts of Poland. The common tick individuals were gathered in the years 2008-2009. Questing ticks were sampled by dragging a white woollen flag over lower vegetation at 17 localities within diverse types of habitats: urban recreational green areas (city parks and squares), suburban forests and rural woodlands throughout the investigated regions of Poland. Detection of B. henselae in tested tick specimens was based on PCR amplification of the citrate synthase (gltA) gene, while screening for the presence of B. burgdorferi s.l. DNA was carried out by analyzing fragments of two genes: the flagellin (fla) and outer surface protein A (ospA). A total number of 1,571 I. ricinus ticks were sampled: 865 (55.1%) nymphs, 377 females (24.0%) and 329 males (20.9%). The application of PCR assays revealed that 76 (4.8%) tick samples were B. henselae-positive, B. burgdorferi s.l. DNA was detected in 194 specimens (12.3%), whereas the co-existence of these pathogens was evidenced in 22 tested ticks (1.4%). Furthermore, the occurrence of bartonellae and co-circulation of analysed microorganisms in I. ricinus was affirmed only within adult individuals, while presence of the screened spirochetes was ascertained in both nymphal and adult ticks. It should be stressed that the suburban woods of Warsaw and rural forests in Warsaw County characterized the highest prevalence levels of dual infection with investigated tick-borne pathogens, whereas the lowest co-infection rates were recorded in tick populations inhabiting rural forests in Płock County and forested areas in Korczew-Mogielnica (within the Nadbużański Landscape Park).