This study investigates the antimicrobial activity of six gut-derived class I lantibiotics, four of which are novel, against both human pathogens and gut commensals. The authors used an improved lantibiotic expression platform to produce and purify these lantibiotics, determined their minimal inhibitory concentrations (MICs), and profiled lantibiotic resistance genes in the pathogens and commensals. Structure–activity relationship (SAR) studies with analogs revealed key regions and residues that impact their antimicrobial properties. The findings provide insights into the future development of lantibiotic-based therapeutics and food preservatives.This study investigates the antimicrobial activity of six gut-derived class I lantibiotics, four of which are novel, against both human pathogens and gut commensals. The authors used an improved lantibiotic expression platform to produce and purify these lantibiotics, determined their minimal inhibitory concentrations (MICs), and profiled lantibiotic resistance genes in the pathogens and commensals. Structure–activity relationship (SAR) studies with analogs revealed key regions and residues that impact their antimicrobial properties. The findings provide insights into the future development of lantibiotic-based therapeutics and food preservatives.