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Probiotics are increasingly popular, currently. Probiotics have been described with the ability to treat many disorders of the gastrointestinal tract (GIT) such as irritable bowel syndrome (IBS) and Crohn’s disease. Types of probiotics include bacterial strains fromLactobacillusandBifidobacterium. Probiotics can restore balance to gut microbiota by outcompeting pathogenic bacteria for nutrients and secrete antimicrobials to eliminate these bacterial pathogens. However, the viability of most advertised probiotics lose their potency due to being freeze dried into powders during storage or for consuming. Many probiotics become ineffective and produce lower CFUs while traversing through the gastric acids of the digestive system. For these reasons, this study sought to enhance the antimicrobial response of a highly potent probiotic known asBacillus subtilis. B. subtilishas been used to treat many disorders of the gut and secrete many antimicrobials lethal for pathogenic microbes.B. subtiliswas genetically modified to express CRISPR-Cas9 nuclease deletion of theaccAgene (ΔB.subtilismutants), which inhibits expression of an essentialaccAgene a part of the fatty acid synthesis (FAS) metabolic pathway. The CRISPR-Cas9-accA ΔB.subtilismutants were co-cultured withV. harveyiandE. Coli. Bacterial growth, biofilm formation, antimicrobial activity, and antibiotic resistance were quantified. It was found thatΔB.subtilismutants co-cultured withV. harveyiandE. Colilessened bacterial growth, amplified biofilm withV. harveyi, reduced biofilm formation ofE. Coli, the co-cultures with the mutants lacked antimicrobial activity, and increased the antibiotic resistance ofV. harveyiandE. Coli. It can be concluded that there is an immense potential for using genetically engineered probiotic strains to enhance the antimicrobial activity ofB. subtilis, which can amplify the reduction of pathogenic bacteria. However, the safety and frugality of usingB. subtilisas a probiotic requires further consideration.