This protocol describes a detailed process of antibacterial (Streptococcus mutans UA159) activity by nanometer-sized iron sulfide (nFeS). Streptococcus mutans has long been considered to be a critical factor in caries-causing plaque development. The pathogens are enriched in 3D matrix of extracellular polymeric substances and lead to dental caries by producing highly acidic niches. We find that nano-iron sulfide improves the killing efficacy of Streptococcus mutans UA159 by more than 500 times compaired with garlic-derived organosulfur compounds. Here the Fe3O4 nanoparticles and nFeS( Cys-nFeS, GSH-nFeS, NaHS-nFeS, Cyss-nFeS, and DADS-nFeS ) converted from different organosulfur sources were used to evaluate the antimicrobial (Streptococcus mutans UA159) activity within 30 minutes.