The present study employed functional near-infrared spectroscopy (fNIRS) to investigate the effects of virtual companionship on prefrontal cortical activation patterns during mindfulness practice, and to examine whether virtual companionship can enhance post-mindfulness psychophysiological states. Sixty healthy college students were recruited and randomly assigned to either the control group (n = 30) or the experimental group (n = 30). The experimental procedure lasted 20 min, with both groups completing a 5-min guided mindfulness breathing exercise. In the experimental group, an empathetically responsive virtual companion was presented concurrently during the practice. Changes in oxygenated hemoglobin (HbO) concentrations in the prefrontal cortex were measured using fNIRS, while mindfulness state was assessed using the Toronto Mindfulness Scale. The virtual companionship group showed higher post-test TMS scores than controls, F(1,58) = 51.50, p < 0.001, η2 = 0.47. All eight functional connections differed significantly after FDR correction; six were higher and two lower in the virtual companionship group, with FPA_R–PSC_L showing the largest difference (t = 2.90, FDR-p = 0.01). In the virtual companionship group, fALFF was higher in Channel 23 (t(55) = − 2.38, p = 0.02) but lower in Channels 46 (t(55) = 2.02, p = 0.05) and 60 (t(55) = 2.05, p = 0.05). This study provides fNIRS-based physiological evidence supporting the enhancing effects of virtual companionship on mindfulness and demonstrates that virtual companionship can effectively modulate mindfulness-induced patterns of local brain activation and global network integration. This study was not preregistered.