This study investigated the effects of nighttime lighting conditions and stress on the affective appraisal of a virtual environment (VE). The effective application of VEs in emotionally intense simulations requires precise control over their characteristics that affect the user's emotions and behavior. It is known that humans have an innate fear of darkness, which increases after exposure to stress and extrapolates to ecologically valid (immersive) VEs. This study investigated if the simulated level of illumination determines the affective appraisal of a VE, particularly after stress. Participants explored either a daytime or a nighttime version of a VE, after performing either an acute psychosocial stress task (Trier Social Stress Test, or TSST) or a relaxing control task. The affective qualities of the VE were appraised through the Russel and Pratt semantic questionnaire on the valence and arousal dimensions. Distress was assessed through free salivary cortisol, the state self-report scale from the Spielberger State-Trait Anxiety Inventory (STAI), and heart rate. In addition, memory for scenic details was tested through a yes-no recognition test. Free salivary cortisol levels, heart rates, and scores on the STAI all indicate that participants who were subjected to the stress task indeed showed signs of distress, whereas participants in the control group showed no signs of stress. The results of the semantic questionnaire and the recognition test showed no significant overall effect of time-of-day conditions on the affective appraisal of the VE or on the recognition of its details, even after prior stress. The experiences of users exploring the VE were not affected by the simulated lighting conditions, even after acute prior stress. Thus, lowering the illumination level in a desktop VE is not sufficient to elicit anxiety. Hence, desktop VE representations are different from immersive VE representations in this respect. This finding has implications for desktop VE representations that are deployed for serious gaming and training purposes.