The rising prevalence and transmission of tick-borne pathogens (TBPs) represent a significant public health issue all over the world, including in Italy. The region of Lombardy, in Northern Italy, is particularly endemic for TBPs. While evidence indicates a high exposure risk to infected ticks, there is a lack of data on ticks collected from humans. Therefore, a long-term surveillance programme was implemented from 2018 to 2023 to identify selected pathogens in ticks retrieved from humans. The presence of Rickettsia spp., Borrelia burgdorferi sensu lato complex, Francisella spp., Coxiella burnetii and tick-borne encephalitis virus was evaluated. A total of 2,453 ticks were collected and most belonged to the species Ixodes ricinus (93.5 %
n = 2,293). Additionally, Ixodes spp. (n = 35), Ixodes hexagonus (n = 7), Rhipicephalus sanguineus (sensu lato) species complex (n = 16), Dermacentor spp. (n = 5), Dermacentor reticulatus (n = 7) and Dermacentor marginatum (n= 1) were identified. The nymph stage was the prevalent life stage at retrieval (n = 1,778), followed by adults (n = 443) and larvae (n = 134). Overall, 18.7 % (459/2,453) of ticks were infected with at least one pathogen and co-infections were found in 6.3 % (29/459) of the positive ticks. In the examined sample, the frequency of infection in adult ticks (90/443
20.3 %) was higher than that observed in nymphs (335/1778
18.8 %). The most frequently detected pathogens were Borrelia burgdorferi s.l. (10.1 %) and Rickettsia spp. (10 %). Interestingly, Francisella tularensis and Coxiella burnetii were found to be of low prevalence, suggesting a lower exposure of humans to these diseases, despite still posing a serious threat to human health due to their pathogenicity. This study raises awareness about the potential risk of tick-borne diseases in Northern Italy.