The study examined the relationship between alcohol consumption during pregnancy, maternal microbiota, and fetal alcohol spectrum disorder (FASD) diagnosis in infants. The research included 207 fecal samples and 28 vaginal swabs from pregnant women in the Western Cape of South Africa. Results showed that higher alcohol use disorder scores (AUDIT) were associated with lower diversity of maternal gut microbiota. Gut microbiota diversity was also reduced in mothers whose infants were diagnosed with FASD. Analysis suggested depletion of gram-positive bacteria with alcohol use and impaired one-carbon metabolism in mothers with FASD-diagnosed children. Vaginal microbiota was not associated with alcohol consumption or FASD diagnosis. The study suggests that alcohol is associated with maternal gut microbiota disruption with potential consequences for FASD development in exposed infants.