To screen for Limosilactobacillus reuteri strains with excellent safety profiles and high antimicrobial activity, this study systematically evaluated the biosafety, carbohydrate metabolic characteristics, and antimicrobial potential of Limosilactobacillus reuteri GZMV125, a strain previously isolated and preserved in our laboratory, by integrating whole-genome analysis with multidimensional experimental validation, thereby exploring the genetic basis underlying its functional properties. Genomic analysis revealed that the strain harbors one circular chromosome and one plasmid. No critical virulence factors were identified, while a type Ⅲ polyketide synthase gene cluster was detected. Safety assessments confirmed that the strain exhibits no hemolytic activity, and its antibiotic susceptibility profile meets the safety criteria for probiotic strains, indicating favorable biosafety characteristics. Antimicrobial assays demonstrated that its cell-free fermentation supernatant exhibits significant inhibitory effects against a broad range of foodborne pathogens, with the strongest inhibition observed against Listeria monocytogenes. Carbohydrate metabolism studies showed that the strain can efficiently utilize diverse carbon sources, including glucose and xylose, and that its antimicrobial activity is regulated by carbohydrate availability. Xylose was identified as the optimal carbon source for inducing the strongest antimicrobial activity, which may be associated with increased viable cell density and enhanced acid production capacity resulting from carbohydrate utilization. These findings established a potential association among “genomic metabolic potential-carbohydrate utilization phenotype-antimicrobial function.” In conclusion, Limosilactobacillus reuteri GZMV125 exhibits excellent safety, broad-spectrum antimicrobial capability, and strong adaptability in carbohydrate utilization, demonstrating considerable potential for development as a probiotic formulation or food fermentation starter culture. This research provides a scientific basis and theoretical support for its food-grade industrial application.