In order to assess the seroprevalence of Borrelia burgdorferi in Romania and to define associated risk factors, a cross-sectional, observational study was performed in 13 districts during 1999. Sera from healthy blood donors (1598) and from forestry workers (1048) were tested. A two-step testing strategy was used in which sera were tested for anti-B. burgdorferi antibodies by a commercially available passive hemagglutination assays (PHA). All PHA positive sera were then evaluated by Western blot IgG. Demographic data regarding age, sex, profession, work place/residence, duration of employment (forestry workers), animals in the environment, and tick bites history were collected using a questionnaire. Data obtained from serological study were matched with that obtained from the questionnaire. The seroprevalence of B. burgdorferi in blood donors was of 4.3% (range 1.4–8.7%) and 9.3% (range 2.8–31.7%) in forestry workers. Seroprevalence was higher in forestry workers with a tick bite history (10.7 vs. 4.3%, p < 0.05). The highest seroprevalence in blood donors (8.7%) was noted in Maramures, a northern district of the country, whereas in forestry workers the highest seroprevalence (31.7%) was observed in a western district (Arad), where a previous study in entomology has demonstrated the highest density of Ixodes ricinus ticks in Romania.