This study aimed at comparing the fecal microbiota and metabolome of 26 healthy subjects before (HS) and after (HSB) two months of diet intervention based on the administration of durum wheat flour and whole-grain barley Pasta containing the minimum recommended daily intake (3 g) of barley β-glucans. Metabolically active bacteria were analyzed through pyrosequencing of the gene 16S rRNA and community-level catabolic profiles. Pyrosequencing data showed that Clostridiaceae (Clostridium orbiscindens, Clostridium sp.), Roseburia hominis, and Ruminococcus sp. increased while other Firmicutes and Fusobacteria decreased in HSB compared to HS fecal samples. Community-level catabolic profiles were the lowest in HSB. Compared to HS, cultivable lactobacilli increased in HSB fecal samples while Bacteroides, Porphyromonas and Prevotella, Enterobacteriaceae, total coliforms, and Pseudomonas, Alcaligenes and Aeromonas decreased. Metabolome analyses were performed using amino acid analyzer and gas-chromatography mass spectrometry-solid-phase micro-extraction. A marked increase of short chain fatty acids (SCFA) such as 2-methyl-propanoic acid, acetic, butyric and propionic acids was found in HSB with respect to HS fecal samples. Durum wheat flour and whole-grain barley Pasta containing 3% of barley β-glucans appeared to be effective in the modulation of the composition and the metabolic pathways of the intestinal microbiota, leading to an increased level of SCFA.