Background Unilateral neglect is common among right-hemispheric stroke individuals and also concerns the auditory modality. Prism adaptation can improve auditory extinction during a dichotic listening task, but its effect during an ecological task has not been studied. Objective The main objective was to evaluate whether lateralized cueing before and after prism adaptation improved virtual spatial navigation of stroke individuals with visual and auditory unilateral neglect. Secondary objectives were to assess spatial memory and obtain a better understanding of the mechanism of the cueing treatment by using an eye-tracker. Methods We included 22 stroke individuals with left visual and auditory neglect, 14 individuals without neglect, and 12 healthy controls. After a familiarization task, participants underwent 3 evaluation sessions. Participants were first passively shown a path that they had then to actively reproduce by using a joystick. A path with lateralized beeping sounds indicating direction and a path without any sounds were followed in a randomized order. After prism adaptation, the participants followed a third path with lateralized beeping sounds. The time of navigation and number of trajectory mistakes were recorded. After navigation, spatial memory was assessed. Additionally, an eye-tracker was used during the navigation period. Results The navigational performance of participants with neglect was significantly better with than without auditory cues, especially after prism adaptation. With auditory cues, participants without neglect reached the navigational performance of healthy controls. The spatial memory of individuals with neglect was significantly lower with auditory cues. Eye-tracking analyses showed that participants with neglect made more saccades and looked longer at the right-square angles in the absence of auditory cues. Conclusions This study demonstrates the positive effect of auditory cues in virtual spatial navigation of individuals with visual and auditory neglect and the potentiation of the help of cues after prism adaptation.< Réduire