The goal was to clarify the audiologic aspects and causes of congenital hearing loss in children who failed universal neonatal hearing screening. A prospective analysis of 170 consecutive records of neonates referred to a tertiary center after universal neonatal hearing screening failure, between 1998 and 2006, was performed. The data presented here represent the equivalent of approximately 87000 screened newborns. The screening results were validated with a clinical ear, nose, and throat examination and electrophysiological testing, including diagnostic auditory brainstem response, automated steady state response, and/or behavioral testing. A diagnostic evaluation protocol for identification of the cause of the hearing loss was also implemented, in collaboration with the departments of genetics and pediatrics. Permanent hearing loss was confirmed in 116 children (68.2%). Bilateral hearing loss was diagnosed in 68 infants (58.6%) and unilateral hearing loss in 48 infants (41.4%). Median thresholds for the neonates with confirmed hearing loss were severe in both unilateral and bilateral cases, at 70 dB nHL and 80 dB nHL, respectively. In 55.8% of those cases, no risk factors for hearing loss were found. In 60.4%, the initial automated auditory brainstem response diagnosis was totally in agreement with the audiologic evaluation results. In 8.3% of the cases, however, a unilateral refer result was finally classified as bilateral hearing loss. An etiologic factor could be identified in 55.2% of the cases. Of the causes identified, a genetic mechanism was present in 60.4% of the cases, peripartal problems in 20.8%, and congenital cytomegalovirus infection in 18.8%. An etiologic factor could be identified for nearly one half of the children with confirmed congenital hearing loss referred through a universal hearing screening program.