The aim of this cross-sectional study was to assess the seroprevalence of antibodies against varicella zoster (VZV), herpes simplex type 1 (HSV-1) and type 2 (HSV-2), hepatitis B (HBV) and hepatitis A (HAV) viruses in adolescents (14-17 years of age) in Madrid, Spain. At the study visit, demographic data and blood samples were obtained. The enzyme linked immunosorbent assay (ELISA) method was used to assess the presence of anti-VZV, anti-HSV-1, anti-HSV-2, anti-HBc and anti-HAV antibodies. A total of 1191 serum samples were collected. Mean age (SD) and male/female ratio of the study population were 15.3 (1.1) years and 0.9, respectively. Seroprevalences obtained were as follows: anti-VZV (94%), anti-HSV-1 (46%), anti-HSV-2 (5%), anti-HBc (3%) and anti-HAV (5%). These data show that Spanish adolescents should be considered a target group for prevention programmes against HSV-2, HBV and HAV infections.