Abstract: Antimicrobial resistance (AMR) is a global healthcare issue that threatens lives by enabling pathogens to evade currently available treatment options, making the discovery or development of new therapies critical. An alternative to traditional antimicrobial drug development is surveying environmental microorganisms that produce novel antimicrobial agents. In this study, an unknown organism isolated from a shoe exhibited antimicrobial activity on Sabouraud Dextrose Agar (SDA). The classification of the organism was based on cultural characteristics, staining (simple, Gram, and endospore stains), biochemical testing, and genetic analysis. The unknown organism was identified as a Gram-positive endospore-forming bacillus based on staining. Polymerase chain reaction (PCR) using universal 16S rRNA primers amplified a 1500-base-pair DNA fragment, and Nucleotide BLAST analysis of the PCR DNA fragment identified the unknown organism as a member of the Bacillus genus. Biochemical tests produced a metabolic profile that most closely matched the unknown organism to Bacillus velezensis. Cross-streaking with Gram-negative and Gram-positive bacteria displayed potential inhibition for Enterobacter cloacae ATCC 23355, Bacillus megaterium ATCC 14581, Staphylococcus aureus ATCC 25923, and Staphylococcus saprophyticus ATCC 15305, suggesting antimicrobial activity. Future research includes verifying the identity of the unknown organism and the isolation and characterization of the antimicrobial agent.