In Europe, Ixodes ricinus ticks are vectors of many emerging pathogens, including Borrelia burgdorferi sensu lato (sl), Anaplasma phagocytophilum, spotted fever group Rickettsia sp., Babesia sp., and very likely Bartonella sp. In this study, we looked for the presence of DNA of these microorganisms in 572 ticks from two forests in the west of France. DNA extraction and polymerase chain reaction (PCR) amplification were performed on individual nymphal, male, and female I. ricinus ticks. Amplification from 1 tick among the 572 samples (0.2%) resulted in PCR products with Bartonella-specific primers. Sequence analysis of the amplified fragment did not lead to species identification. Two ticks (0.3%) carried A. phagocytophilum-specific DNA. Eight ticks (1.4%) were positive with spotted fever group Rickettsia-specific primers, and all PCR fragments were related to Rickettsia helvetica. Thirty-five ticks (6.1%) were positive with B. burgdorferi sl-specific primers; the sequences were all related to Borrelia garinii or Borrelia afzelii, except one that was related to Borrelia carolinensis, a newly described species never reported in Europe so far. Thirty-five ticks (6.1%) carried Babesia sp. DNA. Female adults were more infected by B. burgdorferi sl than male adults. The prevalence of B. burgdorferi sl and Babesia sp. was significantly different between the two forests, with a higher prevalence of B. burgdorferi sl in ticks from the forest of Princé and a higher prevalence of Babesia sp. in ticks from the forest of Gâvre. To our knowledge, this is the first study that has detected all five pathogens in questing I. ricinus in the west of France and the first report of B. carolinensis DNA in ticks in Europe.