This study investigated the onset and development of the immune response to Borrelia burgdorferi infection in 30 Italian patients with culture-confirmed Lyme Borreliosis in the stage of erythema migrans (EM). All patients received antimicrobial treatment when entering the study and were prospectively evaluated monthly for up to 30 days after enrolment. A total of 60 serially collected serum samples were tested by using two different commercial enzyme-linked immunosorbent assays (ELISAs): Anti-Borrelia plus VlsE ELISA, Euroimmun, and the synthetic peptide-based ELISA, Quick ELISA C6, Immunetics. Sixty-five potentially cross-reacting sera were also tested. Anti-Borrelia plus VlsE ELISA IgG was far more sensitive than Quick ELISA C6 (56.6% and 33.3%, respectively). Moreover, considering that 17 additional sera from the first bleeding group of Lyme disease patients were IgM positive when tested by Anti-Borrelia plus VlsE IgM, the sensitivity of Anti-Borrelia plus VlsE as a whole system rose to 85.0%. Nevertheless, due to the specificity values of Anti-Borrelia plus VlsE ELISA identified in this study (98.5% for IgG and 78.5% for IgM), the need of a confirmatory test for the diagnosis of Lyme disease remains. All the sera were also tested by two different commercial Western Blot (WB) assays: Euroline-WB against Borrelia, Euroimmun, and Qualicode B. burgdorferi WB, Immunetics, in comparison with a multispecies "home made" WB. Performances of the three WB methods for the detection of IgM were very similar. On the contrary, these WBs performed with different values of sensitivity and specificity when IgGs were evaluated. The most sensitive method was the "home-made" WB IgG (71.7%), followed by the Euroline-WB IgG against Borrelia (68.3%). Qualicode B. burgdorferi WB IgG demonstrated to be only 26.6% sensitive. Both "home-made" WB IgG and Qualicode B. burgdorferi WB IgG were 100% specific, whereas Euroline-WB IgG against Borrelia scored 12 cross-reacting samples as borderline, showing a specificity value of 80.0%.