This study describes a novel workflow for culturing and characterizing the human intestinal microbiota, which includes targeted phenotypic culturing linked to whole-genome sequencing, phylogenetic analysis, and computational modeling. The approach reveals that a significant proportion of intestinal bacteria are culturable, and at least 50–60% of the bacterial genera from the intestinal microbiota of healthy individuals produce resilient spores, facilitating host-to-host transmission. The study isolates 137 bacterial species, including 45 candidate novel species, and identifies 66 conserved genes linked to ethanol-resistance and sporulation. These findings challenge the notion that many intestinal bacteria are 'unculturable' and highlight the importance of spore-forming bacteria in microbiota persistence and inheritance. The workflow enables large-scale culturing, archiving, genome sequencing, and phenotyping of novel bacteria, contributing to a better understanding of the human intestinal microbiota.This study describes a novel workflow for culturing and characterizing the human intestinal microbiota, which includes targeted phenotypic culturing linked to whole-genome sequencing, phylogenetic analysis, and computational modeling. The approach reveals that a significant proportion of intestinal bacteria are culturable, and at least 50–60% of the bacterial genera from the intestinal microbiota of healthy individuals produce resilient spores, facilitating host-to-host transmission. The study isolates 137 bacterial species, including 45 candidate novel species, and identifies 66 conserved genes linked to ethanol-resistance and sporulation. These findings challenge the notion that many intestinal bacteria are 'unculturable' and highlight the importance of spore-forming bacteria in microbiota persistence and inheritance. The workflow enables large-scale culturing, archiving, genome sequencing, and phenotyping of novel bacteria, contributing to a better understanding of the human intestinal microbiota.