Ticks are important vectors of many pathogens in Europe, where the most impactful species is Ixodes ricinus. Recently, the geographical distribution of this tick species has been expanding, resulting in an increased risk of human exposure to tick bites. With the present study, we aimed to screen 350 I. ricinus specimens collected from humans and wild animals (mainly ungulates), to have a broader understanding of the tick-borne pathogens circulating in the Lombardy region, in northern Italy. To do so, we took advantage of a high-throughput real-time microfluidic PCR approach to screen ticks in a cost-effective and time-saving manner. Molecular analysis of the dataset revealed the presence of four genera of bacteria and two genera of protozoa: in ungulates, 77 % of collected ticks carried Anaplasma phagocytophilum, while the most common pathogen species in ticks removed from humans were those belonging to Borrelia burgdorferi sensu lato group (7.6 %). We also detected other pathogenic microorganisms, such as Rickettisa monacensis, Rickettsia helvetica, Neoehrlichia mikurensis, Babesia venatorum, and Hepatozoon martis. Besides, we also reported the presence of the pathogenic agent Borrelia miyamotoi in the area (1.4 % overall). The most common dual co-infection detected in the same tick individual involved A. phagocytophilum and Rickettsia spp. Our study provided evidence of the circulation of different tick-borne pathogens in a densely populated region in Italy.