Abstract Background New primary preventive therapeutic strategies for atopic dermatitis (AD) are needed. Atopic diseases are associated with disrupted gut microbial balance in early life, suggesting that optimizing microflora through intervention could improve health. Prebiotics, which are immunomodulatory sugars, promote gut microbiota diversity. Most clinical trials focus on improving postnatal infant gut colonization, but prenatal life is crucial for establishing tolerance mechanisms. Preclinical studies indicate that maternal intake of galacto-oligosaccharide (GOS)/inulin prebiotics decreases the risk of food allergy in offspring. Objectives To determine whether antenatal prebiotic intake prevents AD in children at high risk of atopy. Methods The multicentre double-blind randomized PREGRALL clinical trial ran from February 2018 to April 2023 (ClinicalTrials.gov NCT03183440). The follow-up period extended from 20 weeks of amenorrhoea in pregnant women to their infants reaching 1 year of age. Women with a physician-diagnosed history of atopy (asthma, allergic rhinitis, AD or food allergy) were selected for inclusion. The women were randomized to daily prebiotic GOS/inulin (n = 188) or placebo (maltodextrin; n = 188 participants) intake from 20 weeks’ gestation until delivery. The first outcome was the occurrence of AD at 1 year in children at risk of the disease. Secondary endpoints included AD severity, quality of life, prebiotic tolerance and the prevalence of other atopic diseases. Results Of 376 pregnant women included in the trial, prebiotic supplementation did not prevent AD at 1 year (intention-to-treat population odds ratio 1.01, 95% confidence interval 0.59–1.74; P = 0.97) or reduce disease severity in their children. Subgroup analyses by breastfeeding status or delivery mode revealed no differences. No effects on allergen sensitization or food allergies were found. Conclusions We found no evidence that maternal intervention with prebiotics protects against AD at 1 year of age in infants at risk of allergic diseases.