Aims: To develop a microassay method to detect antimicrobials produced by spore-forming bacteria, thus speeding up the discovery of new antimicrobials. Methods and Results: Environmental isolates were grown in 96-well plates, to allow production of antimicrobial agents, then treated with lysozyme and heated sequentially. Lysozyme heat treatment inhibited or prevented spore-to-cell transformation, thus eliminating interference from spore outgrowth while detecting antimicrobials by indicator bacteria. Supplementation of the indicator strain medium with 2,3,5-triphenyltetrazolium chloride, as a vital stain, made it easy to rapidly differentiate between antimicrobial-deficient (indicator growth) and antimicrobial-containing (no growth) wells. The method was used to rapidly screen 657 bacteria isolated from eight soil samples. Results revealed 46 Bacillus sp. producing antimicrobials against Listeria sp., and a Bacillus sp. producing antimicrobial(s) against Escherichia coli. Conclusions: A microassay method was successfully developed and implemented to screen and detect antimicrobial agents from spore-forming, in addition to nonspore-forming, bacteria. Significance and Impact of the Study: Antimicrobials are needed to combat antibiotic- and preservative-resistant bacteria. Spore-forming bacteria are prolific producers of antimicrobials. This assay will speed the discovery of antimicrobials from spore-forming bacteria; these new antimicrobials are urgently needed in food and medicinal applications.