The current project is a preliminary attempt at assessing the potential of a three-dimensional virtual reality Stroop task (VRST) for investigation of supervisory attentional processing. The VRST was compared with paper-and-pencil and computer automated (two-dimensional) versions of the Stroop. Psychophysiological measures were also used to assess varying levels of user arousal. The three versions of the Stroop task were conducted with a sample of 50 healthy university students, between the ages of 18 and 28 years, which included equivalent distributions of men and women from ethnically diverse populations. No significant differences were found for age, gender, or education. Results indicated that the typical Stroop effect pattern found in the Delis-Kaplan Executive Function System (D-KEFS) and Automated Neuropsychological Assessment Metrics (ANAM) occurs in the VRST; the complexity condition of the VRST offers potential for assessment of exogenous and endogenous attentional processing; the two single-item presentation formats of the Stroop are comparable; there is a psychophysiological difference in arousal between low- and high-threat zones; and while there is a learning effect present in the VRST across conditions, it is limited by the arousal conditions in the high-threat zones. Additionally, no negative side effects were associated with use of the virtual environment. We conclude that the VRST has the potential to offer a novel approach to assessment of supervisory attentional processing within an ecologically relevant environment.