Multidrug-resistantEnterococcus faeciumrepresent a major concern due to their ability to thrive in diverse environments and cause life-threatening infections. While antimicrobial resistance and virulence mechanisms have been extensively studied, the contribution of bacteriocins toE. faecium’s adaptability remains poorly explored.E. faecium, within the Bacillota phylum, is a prominent bacteriocin producer. Here, we developed a tailored database of 76 Bacillota bacteriocins (217 sequences, including 40 novel bacteriocins) and applied it to uncover bacteriocin distribution patterns in 997 quality-filteredE. faeciumandEnterococcus lactis(formerE. faeciumclade B) genomes. Curated using computational pipelines and literature mining, our database demonstrates superior precision versus leading public tools in identifying diverse bacteriocins. Distinct bacteriocin profiles emerged betweenE. faeciumandE. lactis, highlighting species-specific adaptations.E. faeciumstrains from hospitalized patients were significantly enriched for bacteriocins asentA,bac43,bacAS5 andbacAS11. These bacteriocins strongly associated with antibiotic resistance, particularly vancomycin and ampicillin, and Inc18rep2_pRE25-derivative plasmids, classically associated with vancomycin resistance transposons. Our integrated genomic and epidemiological analysis elucidates meaningful connections between bacteriocin determinants, antimicrobial resistance, mobile genetic elements, and ecological origins inE. faecium. This work significantly expands the knowledge on the understudied bacteriocin diversity in opportunistic enterococci, revealing their contribution to environmental adaptation. Further characterization of strain-level bacteriocin landscapes could inform strategies to combat high-risk clones. Overall, these insights provide a framework for unravelling bacteriocins’ therapeutic and biotechnological potential.