The rise in antimicrobial resistance underscores the urgent need for novel antimicrobial compounds. Bacteria, including Clostridium botulinum, offer a promising natural reservoir for such discoveries. While C. botulinum is best known for its neurotoxin production, its potential as a source of antimicrobial compounds is largely unexplored. This study integrates next-generation sequencing (NGS), bioinformatics, and antimicrobial assays to identify novel antimicrobial compounds, focusing on bacteriocins—ribosomally synthesized and post-translationally modified peptides (RiPPs) targeting closely related species. The nontoxic C. botulinum HA5 strain exhibited antimicrobial activity against clostridial species, including toxigenic C. botulinum, C. difficile, and C. perfringens. Bioinformatics revealed multiple biosynthetic gene clusters (BGCs). Knockout experiments identified a spontaneous mutant lacking antimicrobial activity due to the loss of a plasmid (pHA5) encoding the botocidin biosynthetic gene cluster (a ranthipeptide or sactipeptide). This confirmed the role of botocidin in antimicrobial activity. The botocidin BGC comprised two operons: one encoding the core peptide, modification enzymes, an ABC transporter, and proteases, and the other, oriented oppositely, encoding an MFS transporter and a potential modification enzyme. The Spo0A transcription factor regulated the production operon, while the MFS transporter conferred immunity without affecting production. Botocidin production was confirmed via TCA precipitation and LC-MS, revealing peptide masses consistent with the predicted compound. Stability tests showed botocidin's heat stability (1 h at 90 °C), pH stability (pH 4–9), moderate UV sensitivity, and protease sensitivity. Time-lapse microscopy revealed bactericidal activity, causing cell lysis by targeting the membrane, as confirmed by propidium iodide staining. While botocidin did not inhibit spore germination, it blocked spore outgrowth sporestatically, as shown by plate counts and microscopy. These findings identify botocidin as the active antimicrobial in C. botulinum HA5, highlighting its potential to address antimicrobial resistance.