Neoehrlichia mikurensis is a new emerging tick-borne Gram-negative bacterium, belonging to the family Anaplasmataceae, the main vector of which in Europe is the tick Ixodes ricinus. N. mikurensis is responsible for neoehrlichiosis, occurring mostly in patients with underlying diseases. In the present study, a total of 348 I. ricinus and Dermacentor reticulatus ticks collected in north-eastern Poland were analyzed for the prevalence of N. mikurensis. A total of 140 questing ticks (124 of I. ricinus ticks and 16 D. reticulatus) collected with the flagging method and 208 ticks (105 and 103 I. ricinus and D. reticulatus, respectively) removed from dogs were selected for the study. cDNA (questing ticks) and total DNA (questing and feeding ticks) were analyzed by qPCR targeting the 16S rRNA gene of N. mikurensis. Positive samples were further analyzed by nested PCR and sequencing. The prevalence differed between ticks collected from vegetation (19.3%; 27/140) and ticks removed from dogs (6.7%; 14/208). The presence of the pathogen in questing and feeding D. reticulatus ticks was proven in Poland for the first time. In summary, our research showed that infections of ticks of both the most common tick species I. ricinus and D. reticulatus in north-eastern Poland are present and ticks collected from urban areas were more often infected than ticks from suburban and natural areas. The detection of N. mikurensis in I. ricinus and D. reticulatus ticks from north-eastern Poland indicates potential transmission risk for tick-bitten humans at this latitude.