Ixodid ticks are competent vectors for multiple pathogens, several of which cause infections in human. The medical importance of tick-borne pathogens is well known, yet unanswered questions remain regarding the occurrence of pathogens such as Rickettsia spp., Anaplasma phagocytophilum, and Candidatus Neoehrlichia mikurensis in questing ticks in Romania. Our objectives were to identify three emerging tick-borne zoonotic pathogens in eastern Romania, to assess their prevalence, establish co-infection rates, and to compare infection levels of selected pathogens in questing ticks collected from one suburban area in the city of Iai and one forested area located in the Danube Delta Biosphere Reserve. We collected 490 questing nymphs or adult ticks (467 Ixodes ricinus, 4 Dermacentor reticulatus, and 19 Haemaphysalis punctata). We individually analyzed ticks for the presence of Rickettsia spp., A. phagocytophilum, and C. N. mikurensis. Rickettsia spp. was detected in 9.4% of ticks from both sampling areas. Rickettsia spp. included R. helvetica (n=17 I. ricinus ticks), R. monacensis (n=28 I. ricinus ticks), and R. raoultii (n=1 D. reticulatus). C. N. mikurensis had an infection rate of 4.9% while A. phagocytophilum was detected only in the forested area with a global prevalence of 1.2%. The overall prevalence of ticks infected with at least one pathogen was 15.5%, and 5.3% of infected ticks were tested positives for dual pathogen association. Our study documents the presence of pathogens in questing ticks in the urban recreational areas of Iai and forested areas located in the Danube Delta Biosphere Reserve. Worth mentioning, is the presence of C. N. mikurensis in ticks from eastern Romania, an agent just recently described in Romania, and the existence of co-infections in ticks at a similar prevalence to other European countries.