Investigated were the epidemiologic, ecologic and clinical characteristics of Lyme borreliosis in northwest Croatia. In a seroepidemiologic study, human sera were analyzed by indirect immunofluorescent assay (IFA), and ten out of 134 serum samples were positive for B. burgdorferi antibodies. In a seroepizootiologic study, wildlife and domestic animals were tested by inhibition ELISA. Antibodies to B. burgdorferi were found in nine out of 42 roe deer sera, and in three out of nine hare sera. Sera of wild boars (n = 10), cattle (n = 103), and dogs (n = 13) were negative for antibodies to B. burgdorferi. The presence of Borrelia burgdorferi sensu lato was assessed in ixodes ricinus ticks in the Lyme borreliosis endemic region of northwest Croatia. Ticks (n = 123) were collected at five-different locations and analyzed by polymerase chain reaction (PCR). Borrelia burgdorferi sensu lato DNA was detected in 56 out of 124 ticks (45%). Four genomic groups were identified by genotyping: B. afzelii (n = 26), B. garinii (n = 5), VS116 group (n = 5), and Borrelia burgdorferi sensu stricto (n = 1). Mixed infections of B. afzelii with VS116 group (n = 10), and B. afzelii with Borrelia burgdorferi sensu stricto (n = 1) were also detected. Eight ticks contained B. burgdorferi sensu lato that could not be typed, indicating the possible existence of a specific genomic group of B. burgdorferi sensu lato in northwest Croatia. Sex distribution of Lyme borreliosis patients in northwest Croatia showed a slight preponderance of the female gender and prevalence of the working active age range of 20 to 50 years. The persons who periodically visit the landscape, are most commonly affected, whereas those with an increased risk of tick bites are considerably less frequently involved. The most frequent clinical manifestation of Lyme borreliosis in northwest Croatia is erythema migrans with 46%, followed by neurologic manifestations, particularly peripheral neuritis, with 32%. The rest of clinical manifestations in stage II and III of Lyme borreliosis are very rarely recorded. The presence of B. afzelii and B. garinii in the highest percentage is in agreement with the local occurrence of cutaneous and neurologic manifestations of Lyme borreliosis. The presence of VS116 group in ticks from northwest Croatia in this and other studies in some European countries may indicate that VS116 group is well established in the European ixodes ricinus ticks. The role of the VS116 group in the etiology of Lyme borreliosis remains to be clarified.