Updated project metadata. Brevibacillus sp. SPR20 produced potentially antibacterial substances against MRSA. The synthe-sis of these substances is controlled by their biosynthetic gene clusters. Several mutagenesis methods are used to overcome the restriction of gene regulations when genetic information is ab-sent. Atmospheric and room temperature plasma (ARTP) is a powerful technique to make random mutagenesis for microbial strain improvement. This study utilized an argon-based ARTP to conduct the mutations on SPR20. The positive mutants of 40% occurred. The M27 mutant exhib-ited an increase in anti-MRSA activity when compared to the wild-type, by which the MIC values were 250500 and 500 g/mL, respectively. M27 had genetic stability because it exhibited con-stant activity throughout 15 generations. This mutant had similar morphology and antibiotic susceptibility to the wild-type. Comparative proteomic analysis identified some specific proteins that were upregulated in M27. These proteins were involved in the metabolism of amino acids, cell structure and movement, and catalytic enzymes. These might result in the enhancement of the anti-MRSA activity of the ARTP-treated SPR20 mutant. This study supports the ARTP technology to increase the production of valuable antibacterial agents.