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Despite the significant burden of tick-borne diseases (TBDs), epidemiologic studies are missing, and TBD awareness is low in the Republic of Moldova. Our study is the first to assess the prevalence of the main tick-borne pathogens (TBPs) infecting dogs in this country and associated risk factors. In this cross-sectional, multi-centre study (June 2018–July 2019), blood samples were collected from dogs presenting in veterinary clinics (Chişinău: N = 30) and hosted in public dog shelters (Cahul: N = 42; Chişinău: N = 48). TBPs were assessed by molecular techniques and risk factors by the logistic regression model. <i>Hepatozoon canis</i> was the most prevalent TBP (15.8% [19/120]), followed by <i>Babesia canis</i> (11.7% [14/120]), <i>Anaplasma phagocytophilum</i> (5.8% [7/120]), and <i>Bartonella</i> spp. (0.8% [1/120]). Blood samples tested negative for <i>Borrelia</i> spp., <i>Rickettsia</i> spp., <i>Francisella tularensis</i>, <i>Anaplasma platys</i>, and <i>Ehrlichia canis</i>. Dogs originating from the veterinary clinics had a higher prevalence of <i>A. phagocytophilum</i> infection than those from the shelters (16.6% versus 2.2%, respectively, <i>p</i> = 0.0292; OR: 27.0 [95%CI: 1.4–521.9]). Dogs from Chișinău had a higher prevalence of <i>Hepatozoon canis</i> infection versus those from Cahul (19.2% versus 9.5%, respectively, <i>p</i> = 0.0295; OR: 3.9 [95%CI: 1.1–13.4]). We recommend routine use of acaricides and deworming of dogs to prevent or/and limit TBD spread. Further TBD surveillance studies are needed.