Background. Congenital toxoplasmosis diagnosis in the newborn is a very important issue due to the need for early treatment to prevent future sequels. Aim: To compare available methods at the institution for the diagnosis of congenital toxoplasmosis. Material and Methods: In this study we have evaluated the different diagnostic tests used in 67 congenital exposed newborns, including serological tests, PCR, parasite isolation and molecular characterization. Results: The ISAGA IgM and IgA tests showed sensitivity (Se) of 87 and 91%, respectively, and specificity (Sp) of 100%. When ISAGA IgM and IgA were performed simultaneously, the Se increased to 98% and the Sp was 100%. The presence of IgE contributed to the diagnosis when it was detected in the child’s serum but not in maternal blood. In four congenital infected children the parasite was isolated and genotyped: one was genotype II and the other three were “atypical” genotypes. No parasite was isolated in children without congenital toxoplasmosis. Discussion: Overall, serological tests showed a good diagnostic performance although in one case they were all negative and isolation was the only tool to identify the infection. We conclude that it is essential to use all diagnostic tests in every single exposed child, including if possible, molecular characterization due to its epidemiological implication.