The physiological stress response affects executive functions, such as inhibition, as assessed by the Stroop Color and Word Test. In this study, we investigated the effects of the Virtual Reality Stroop Room (VRSR), a research paradigm assessing these cognitive top-down processes while inducing mild acute stress, on self-reported stress states, heart rate, salivary alpha-amylase, and cortisol. Our sample consisted of 89 participants (52 women; Age: 23.60 ± 3.88 years) and was evenly allocated to the three conditions of the VRSR (regular, time pressure, and rotation). The Stroop Effect, reflected in prolonged processing times and increased errors in the incongruent phase, was observed. Participants reported heightened Distress and Engagement post-experiment, alongside lower Worry, assessed via the Short Stress State Questionnaire. Scores from the Positive and Negative Affect Schedule indicated elevated positive affect and decreased negative affect post-study. With regard to biosignals we found that heart rate was higher in the incongruent phase, compared to the congruent phase and a significant time × condition interaction was observed. Salivary alpha-amylase exhibited a significant time effect. Results for cortisol do not support a uniform response of the hypothalamus-pituitary-adrenal (HPA) axis. In conclusion, the VRSR appears to be a valid measure for executive functions while activating the sympathetic nervous system, but not the HPA axis. Its current implementation induces mild physiological and psychological stress responses, with fewer adverse reactions compared to the Trier Social Stress Test. Future studies should leverage the adaptability of virtual reality applications to refine this research paradigm.