The soil bacterium Rhodococcus is a member of the phylum Actinobacteria and is related to Streptomyces, which is known for its production of many secondary metabolites. Recent genomic investigation of Rhodococcus has uncovered many silent gene clusters that appear to code for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKS) of unknown function. Previous work, showed that Rhodococcus species strain KCHXC3 produces an inhibitory compound in agar culture extracts that displays prominent activity against several Gram positive and Gram negative species including the pathogens Rhodococcus equi, Shigella dysenteriae and Pseudomonas aeruginosa. Using the engineered Rhodococcus transposon vector, pTNR, the goal of this investigation is to screen random mutants of KCHXC3 for strains that no longer produce the inhibitory molecule. A library of 1825 random insertion mutants was produced via electroporation then screened for production of the inhibitory molecule by a disk diffusion assay against Shigella dysenteriae. From this screening, 7 mutants which no longer produce the compound of interest were identified.