Abstract Staphylococcus aureus asymptomatically colonizes a third of the world’s population, and it is an opportunistic pathogen that can cause life threatening diseases. To diagnose S. aureus infections, it is necessary to differentiate S. aureus from the ubiquitous human commensal Staphylococcus epidermidis, which beneficially colonizes the skin of all humans. Efforts are underway to identify volatile biomarkers for diagnosing S. aureus infections, but to date no studies have investigated whether S. aureus and S. epidermidis can be reliably differentiated under a variety of growth conditions. The overall goal of this study was to evaluate the influence of growth medium on the ability to differentiate S. aureus and S. epidermidis based on their volatile profiles. We used headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) to examine the headspace volatiles of S. aureus and S. epidermidis when aerobically grown in four different complex media. We detected 337 volatile features when culturing S. aureus and S. epidermidis in four complex media, termed the staph volatiles, and found only 20%–40% concurrence in the volatiles produced by these two species in any single medium. Using principal components analysis and hierarchical clustering analysis on the staph volatiles, we observed that S. aureus and S. epidermidis clustered independently from each other, and distinctly clustered by growth medium within species. Removing volatiles that are species and/or media-specific from the analysis reduced the resolution between species clusters, but in all models clustering by species overrode clustering by media type. These analyses suggest that, while volatile profiles are media-specific, species differences dominate the staph volatilome. These data enable future investigations into the identification of volatile biomarkers to discriminate staphylococcal pathogens versus commensals, which will improve staph diagnoses and provide insights into the biochemistry of staph infections and immunity.