Bacillus safensis strains have emerged as versatile microbial platforms for bioproduction, combining the benefits of probiotic utility and biocontrol. In this study, we describe the isolation and in-depth characterization of a previously unreported B. safensis strain, LG01. The genome of this strain comprises a circular chromosome encoding 13 secondary metabolite biosynthetic gene clusters, 144 carbohydrate-active enzymes, 2 antibiotic resistance loci, and 1 prophage region, indicative of strong antimicrobial and metabolic capacity. Its protein secretion systems support nutrient acquisition, colonization, quorum sensing, and antibiotic synthesis. Our phenotypic assays confirmed the antifungal and antibacterial activity, proteolytic and cellulolytic functions, and robust biofilm formation of the strain. By performing a comparative genomic analysis, we identified 78 strain-specific genes enriched in the bacteriocin immunity and sporulation pathways. Signals of positive selection in the membrane and transcriptional regulator genes further reflect the adaptive evolution underlying the strain’s ecological fitness. Together, these findings advance our understanding of the genomic features of B. safensis LG01 and highlight its promise as a candidate for biocontrol and probiotic applications.