To screen Limosilactobacillus reuteri strains that combine excellent safety with high antimicrobial activity, this study used the previously isolated and preserved strain Limosilactobacillus reuteri GZMV125（L. reuteri GZMV125）as the research subject. An integrated strategy combining whole-genome analysis with multi-level experimental validation was employed to systematically evaluate its biosafety, carbohydrate- metabolizing characteristics, and antimicrobial potential, and to elucidate the genetic mechanisms underlying these functional traits. Genome analysis revealed that the strain harbors one circular chromosome and one plasmid with a high coding density, lacks key virulence factors, and carries bacteriocin-transport-related genes and a type-III polyketide synthase gene cluster. Safety assays confirmed the absence of hemolytic activity and an antibiotic-susceptibility profile consistent with safe strains, indicating excellent biosafety. Antimicrobial tests showed that the cell-free supernatant significantly inhibited diverse food-borne pathogens, with the strongest effect against Listeria monocytogenes. Carbohydrate-metabolism studies demonstrated efficient utilization of glucose, xylose, and other carbon sources; antimicrobial activity was carbohydrate-dependent, with xylose inducing the highest activity. The core mechanism is carbohydrate-mediated high viable-cell density and strong acid-production capacity, establishing a regulatory link among “genomic metabolic potential–carbohydrate -utilization phenotype–antimicrobial function”. In summary, L. reuteri GZMV125 combines high safety, a broad antimicrobial spectrum, and strong adaptability to carbohydrate utilization, offering great potential for development as a probiotic or food-fermentation starter. This study provides a scientific basis and theoretical support for its food-grade industrial application.