Previous in vitro and in vivo animal studies showed that O₂ and CO₂ concentrations can affect virulence of pathogenic bacteria such as Staphylococcus aureus. The objective of this work was to measure O₂ and CO₂ levels in the vaginal environment during tampon wear using newly available sensor technology. Measurements by two vaginal sensors showed a decrease in vaginal O₂ levels after tampon insertion. These decreases were independent of the type of tampons used and the time of measurement (mid-cycle or during menstruation). These results are not in agreement with a previous study that concluded that oxygenation of the vaginal environment during tampon use occurred via delivery of a bolus of O₂ during the insertion process. Our measurements of gas levels in menses showed the presence of both O₂ and CO₂ in menses. The tampons inserted into the vagina contained O₂ and CO₂ levels consistent with atmospheric conditions. Over time during tampon use, levels of O₂ in the tampon decreased and levels of CO₂ increased. Tampon absorbent capacity, menses loading, and wear time influenced the kinetics of these changes. Colonization with S. aureus had no effect on the gas profiles during menstruation. Taken collectively, these findings have important implications on the current understanding of gaseous changes in the vaginal environment during menstruation and the potential role(s) they may play in affecting bacterial virulence factor production.