Bacteriocins have gained attention as alternative therapeutic agents in pharmaceuticals and are also used as preservatives in the food industry. This study focused on the characterization of bacteriocin-like inhibitory substances (BLISs) produced by a potential probiotic strain Bacillus paranthracis strain MHSD3 and evaluations of their antibacterial activity against Gram-positive and Gram-negative pathogenic strains using disc diffusion method. The inhibitory substances were characterized based on their sensitivity to heat, pH, enzymes, and treatment with organic solvents. Inhibitory activity was observed against Escherichia coli, Staphylococcus aureus, S. saprophyticus and S. epidermidis. The BLIS remained active over different ranges of temperature, pH, enzymes, and solvents, but was sensitive to chloroform. Furthermore, proteomic analysis using liquid chromatography–mass spectrometry (LC–MS) revealed the presence of peptides with potential bacteriocin-like characteristics. These results indicates that the BLIS may be a potential candidate to be used as a therapeutic agent.