The increasing abundance and distribution of hard ticks within Europe have been reported extensively over the last 10–20 years. Tick populations in urban and peri-urban environments are increasingly recognized as significant vectors for a wide array of pathogens that not only threaten the health of domestic animals but also pose a risk to public health. Urban wild animals are suitable hosts for ixodid vectors, and therefore they may play a role as reservoir of vector-borne pathogens. MALDI-TOF MS has recently been proposed as an accurate tool for arthropod identification, and this includes hard ticks’ identification. Urban wildlife is often parasitized with a wide species range of hard ticks. We decided to construct a main spectra library of ixodid ticks. For this purpose, we used specimens recovered from urban wild mammals (hedgehogs, roe deers and foxes) hosted at the Wildlife Hospital of the Veterinary College of Alfort (EnvA), in Paris, France. We succeeded to create an MSP database for the identification of Ixodes hexagonus and I. ricinus. The establishment of an Ixodes MSP bank at EnvA will allow further studies in wildlife or domestic hosts using a method considered faster and cheaper than conventional methods. This approach will increase the possibilities of vector identification by transporting specimens simply in ethanol, and will represent a valuable tool for ecological and epidemiological studies. Furthermore, we have started a preliminary study that aims to contribute to a better knowledge regarding the circulation of tick-borne pathogens in ticks recovered from urban wild mammals from Paris region. The presence of these pathogens in the blood meal ticks engorged on wild animals will provide insights into the role of urban wildlife as reservoir/sentinels for infectious diseases transmissible to domestic animals or to humans. Also, we assessed the circulation of tick-borne pathogens in urban wild mammals by blood sampling and high-throughput molecular screening.