The detection of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in meconium has been investigated recently as an alternative to meconium fatty acid ethyl esters (FAEEs) measurement as an objective estimate of prenatal alcohol exposure, independent of maternal self-reporting. We report the results of the first study conducted to investigate the concentrations of EtG and EtS in meconium from newborns with and without intrauterine exposure to ethanol, defined by questionnaire and meconium FAEEs concentration. FAEEs, EtG, and EtS were quantified by liquid chromatography tandem mass spectrometry in meconium samples obtained from the Arcispedale Santa Maria Nuova, Reggio Emilia, Italy (n = 80) and from the Hospital del Mar in Barcelona, Spain (n = 105). Median EtG and EtS values in meconium from newborns without intrauterine exposure to ethanol varied between 0.100 and 0.140 nmol/g and 0.010 and 0.020 nmol/g in Reggio Emilia and Barcelona samples, respectively. In meconium from newborns with uncertain prenatal ethanol exposure, the EtG median value was 0.160 nmol/g in the Italian cohort and 0.250 nmol/g in the Spanish one. The median EtS concentration was 0.020 in both cohorts. EtG and EtS median values in 5 meconium samples from newborns of heavily drinking mothers were 7.240 nmol/g and 0.033 nmol/g, respectively. A positive cutoff of 2.0 nmol/g for EtG yielded the best sensitivity and specificity (100%) to discriminate for true prenatal exposure to ethanol. It was not possible to establish a proper cutoff for EtS because of the low number of positive samples. Based on our results, meconium EtG can be proposed as an alternate biomarker for intrauterine alcohol exposure. In contrast to the 7 FAEEs, EtG is just one molecule that could be screened in meconium samples from all newborns by a simple, low-cost, easy-to-perform immunoassay, which can be routinely applied in neonatology wards for the early diagnosis of prenatal exposure to ethanol.