Background: The development of gut microbiota in infancy is important in the maturation of the immune system. Deviations in colonization patterns have been associated with allergic manifestations (e.g. eczema), but exact microbiome dysfunctions underlying allergies remain unclear. We studied the gut microbiota of 138 infants at increased risk of developing allergy, participating in a clinical trial investigating the effectiveness of a partially hydrolyzed protein formula supplemented with non-digestible oligosaccharides (pHF-OS) on the prevention of eczema. Objective: The effects of the interventions and breastfeeding on fecal microbiota were investigated. Additionally, we aimed to identify microbial patterns associated with the onset of eczema. Methods: Bacterial taxonomic compositions in the first 26 weeks of life were analyzed using 16S rRNA-gene sequencing. Additionally, fecal pH and microbial metabolites were measured. Results: Fecal microbial composition, metabolites and pH of infants receiving pHF-OS was closer to breastfed infants than to infants receiving standard cow’s milk formula. Infants with eczema by 18 months showed discordant development of bacterial genera of Enterobacteriaceae and Parabacteroides spp. in the first 26 weeks, as well as decreased acquisition of lactate-utilizing bacteria producing butyrate, namely Eubacterium and Anaerostipes spp., supported by increased lactate and decreased butyrate levels at 26 weeks. Conclusions: We showed that a pHF with specific prebiotics modulated the gut microbiota closer to that of breastfed infants. Additionally, we identified a potential link between the microbial activity and onset of eczema, which may reflect a suboptimal implementation of gut microbiota at specific developmental stages in infants at high-risk for allergy.