Persons with cystic fibrosis (CF) are highly susceptible to lung infections caused by the opportunistic pathogens Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA). By age 20, ~16% of CF patients have co-infections with these two bacteria, and this number grows as the patients age1. PA-SA co-infections are associated with worsened clinical outcomes in CF patients, but the reasons are not well understood. One hypothesis is that SA influences the production of PA virulence factors and other chronic infection phenotypes. Previous work in our lab investigated the effects of SA on PA quorum-regulated phenotypes when they are grown as planktonic co-cultures. We are expanding on this result by testing whether SA can influence PA phenotypes without being in direct contact, and without being able to exchange soluble secreted factors. In this study, we hypothesized that SA produces volatile organic compounds (VOCs) that cause changes in PA phenotypes leading to a down-regulation of motility and protease production, and increased antibiotic resistance. To test this hypothesis, we exposed two laboratory strains of PA to the VOCs produced by pre-grown lawns of two strains of SA, and measured PA motility by conducting swarming, swimming, and twitching assays, measuring protease production, as well as antibiotic sensitivity. After exposing PA to a pre-grown lawn of SA, there was a significant difference in some phenotypes compared to controls. There were significant decreases in swarming motility, twitching motility, and protease production, and an increase in a bright green pigment (possibly siderophores) when PA was exposed to SA. The degree of phenotypic alterations was dependent on both the PA strain and the SA strain being tested. Exposure to SA VOCs also altered PA sensitivity to ciprofloxacin, though one strain caused an increase in susceptibility while the other SA strain caused an increase in resistance. These data demonstrate that SA VOCs can influence PA phenotypes in vitro, which may have relevance for CF patients who are co-infected with these two bacteria.