Human cytomegalovirus is a ubiquitous, large enveloped DNA β-herpesvirus that, like other herpesviruses, establishes lifelong latency following primary infection. It is the most frequent cause of congenital, neonatal and early postnatal infections with long lasting sequelae. The aim of the present study was to assess the prevalence of cytomegalovirus among a cohort of newborns and 1-3-month-old children with neurological symptoms, physical retardation, prolonged jaundice, thrombocytopenic purpura and other disabilities. The study was a retrospective cross-sectional analysis of serological screening data for detection of specific anti-cytomegalovirus IgM and IgG in children from Northeastern Bulgaria. Between 2003 and 2015, average prevalences of 18.8% (95% CI: 15.4 to 22.2) for anti-CMV IgM antibodies (suggesting acute infection) and 84.7% (95% CI: 81.6 to 87.8) for anti-CMV IgG antibodies were measured in a total number of 517 samples. The prevalence rate of anti-CMV IgM in 1-3-month-old children was 4-fold higher than that in newborns [25.8% (95% CI: 21.1 to 30.5) and 6.4% (95% CI: 2.9 to 9.9, respectively]. In contrast, no significant difference was found for anti- CMV IgG positivity between newborns and 1-3-month-old infants (84% and 85%, respectively). The data obtained strongly encourage screening of pregnant women for anti-CMV IgG and IgM to avoid transmission of the infection and severe complications of congenital infection.