OBJECTIVE Limited data exist on differences of erythema migrans patients with either positive or negative Borrelia burgdorferi sensu lato skin culture. METHODS We analyzed 252 adult patients with erythema migrans in whom skin biopsy specimen was cultured for the presence of B. burgdorferi sensu lato. Evaluations of epidemiological, clinical, and microbiological findings were conducted at baseline, 14 days, 2, 6, and 12 months after treatment with either doxycycline or cefuroxime axetil. RESULTS One hundred fifty-one (59.9%) patients had positive skin culture (86.9% B. afzelii, 8.0% B. garinii, 5.1% B. burgdorferi sensu stricto) and 101 (40.1%) had negative skin culture. Patients in the culture-positive and culture-negative groups were comparable for the basic demographic, epidemiological, clinical, and laboratory characteristics at presentation. Statistically significantly worse selected treatment outcome parameters in the culture-positive group compared with the culture-negative group were established during follow-up. Treatment failure was documented in two patients who were culture positive and in none in the culture-negative group. CONCLUSIONS Although findings for the pretreatment characteristics were comparable between the erythema migrans skin culture-positive and culture-negative patients, some parameters indicate that borrelia skin culture positivity may predict a less-favorable treatment outcome.