In this study, raising and development of antibody response to Borrelia burgdorferi infection in 66 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans (EM) was investigated. Sixty-two of 66 cultures obtained from biopsies were identified as B. afzelii by PCR. A total of 175 serially collected serum samples were tested by using two different sets of commercial assays: Enzygnost Lyme link VlsE/IgG and Enzygnost Borreliosis IgM (DADE Behring, Marburg, Germany) and LIAISON Borrelia IgG and IgM (Diasorin, Saluggia, Italy). Considering only samples obtained at first presentation when EM was clinically evident, 49/66 patients (72.4%) were IgG or IgM positive by Enzygnost, whereas 33/66 (50.0%) patients were IgG or IgM positive by LIAISON. Taking into account the follow-up period, eight patients sero-converted for IgG or IgM by Enzygnost and four by LIAISON. Similar and very good specificity values were obtained by all methods. Testing sera obtained from blood donors (n = 300) and from patients suffering from some of the most common biological conditions possibly resulting in false-positive reactivity in Lyme disease serology (n = 100) showed that Enzygnost Lyme link VlsE/IgG was the more specific (98.3%), followed by LIAISON Borrelia IgG (96.5%), and considering IgM tests, Enzygnost Borreliosis IgM showed to be 95.3%% specific, whereas the LIAISON Borrelia IgM was 92.8% specific. Recombinant VlsE antigens obtained from all three B.burgdorferi genospecies pathogenic to humans (included in Enzygnost Lyme link VlsE/IgG) greatly improved serodiagnosis of Lyme disease.