Nontargeted metabolomics analyses were used (1) to compare fecal metabolite profiles of healthy breast-fed (BF) and formula-fed (FF) infants before and during in vitro fermentation in batch culture and (2) to evaluate fecal metabolomics in assessing infant diet. Samples from healthy BF (n = 4) or FF (n = 4) infants were individually incubated at 37( °)C in anaerobic media containing 1% (wt/vol) galactooligosaccharides, 6'-sialyllactose, 2'-fucosyllactose, lacto-N-neotetraose, inulin, and gum arabic for up to 6 h, and supernatants were analyzed using GC/MS and LC/MS/MS to assess changes in various compounds. Comparison of over 250 metabolites prior to incubation showed that BF samples contained higher relative concentrations (P ≤ 0.05) of 14 compounds including human milk oligosaccharides and other metabolites presumably transferred through breast feeding (linoelaidate, myo-inositol) (P ≤ 0.05). Conversely, feces from FF infants contained 41 identified metabolites at higher levels (P ≤ 0.05) with many indicative of carbon limitation and protein fermentation. Our data are consistent with the notion that carbon-limited cultures catabolize protein and amino acids to obtain energy, whereas the provision of fermentable carbohydrate creates anabolic conditions relying on amino acids for bacterial growth. Results also suggest that fecal metabolomics can be a useful tool for studying interactions among diet, microbes, and host.