Variovorax paradoxus EPS is a gram-negative rod isolated from the sunflower rhizosphere at CSUSB. Preliminary research has shown that Variovorax paradoxus EPS has anti-staphylococcal activity in liquid and solid co-culture. Anti-staphylococcal activity of Wild type and V. paradoxus EPS 𝚫4519 on 0.5% YE agar with embedded S. aureus AH1710 supports the idea that a soluble molecule is responsible for this activity, as the agar acted as a physical barrier between V. paradoxus EPS and S. aureus colonies. Preliminary genetic analysis of V. paradoxus EPS identified three loci that suitable candidates for the synthesis of a potential anti-staphylococcal small molecule. Preliminary data failed to detect expression at two of the three identified loci and a strain with a mutation at the third locus continues to produce anti-staphylococcal activity. We hypothesize that the microbial agent is expressed at a different locus or loci that have not yet been identified. These gene products are responsible for the synthesis of the microbial agent and are controlled by exposure to Staphylococcus aureus. Optimal growth conditions were identified for V. paradoxus EPS and S. aureus to demonstrate the formation of a zone of inhibition on Tryptic Soy Agar. The use of a V. paradoxus EPS Δ 4519 transposon library at optimal growth conditions allowed us candidate mutants with altered antimicrobial activity phenotypes. We identified 28 insertion sites that resulted in altered antimicrobial activities, which will allow us to identify the genes involved in this biosynthetic pathway.