Ixodes ricinus, the most common tick species in Northern Europe, plays a significant role as a vector of several pathogens, with its geographical distribution expanding in recent years. In Southern Sweden, particularly in Region Skåne County (referred to as Skåne), the favorable climate and landscape conditions support extensive proliferation of I. ricinus. Despite Lyme borreliosis being common in this region and few annual cases of tick-borne encephalitis (TBE) being reported, data on the circulation of tick-borne pathogens (TBPs) remain limited. This study molecularly investigated the presence of Anaplasma phagocytophilum, Babesia spp., Borrelia spp., and TBE virus (TBEV) in I. ricinus ticks (n = 1000). In detail, questing ticks (82 adults and 196 nymphs) were collected from vegetation in forest and meadow areas, while 581, 80 and 8 feeding adults were collected from 39 roe deer, 6 fallow deer and 1 moose, respectively. Additionally, 53 feeding adults were removed from domestic animals (42 from four dogs and 11 from one cat). The molecular analyses detected Anaplasma phagocytophilum, Borrelia spp., and Babesia spp. in 54 %, 24 %, 3.2 % of host-feeding ticks and in 0.40 %, 35 %, 3.6 % of questing ticks, respectively. In detail, for Borrelia and Babesia genera, the following species were detected: Borrelia miyamotoi, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi s.s., Babesia microti and Babesia venatorum. TBEV was not detected. Moreover, the relationship between the feeding duration of the roe deer-collected ticks and their PCR-positivity for Borrelia spp. and A. phagocytophilum was also modeled. The results showed a reduction in the probability of tick infection with Borrelia spp. as attachment time increased, supporting evidence that roe deer serum exerts a borreliacidal effect. This study highlights the presence of several zoonotic TBPs in Skåne, emphasizing the need for a structured monitoring plan and preventive strategies within a One Health framework.