Abstract Background Unilateral spatial neglect (USN), a highly prevalent and disabling post-stroke impairment, has been shown to affect the recovery of locomotor and navigation skills needed for community mobility. We recently found that USN alters goal-directed locomotion in conditions of different cognitive/perceptual demands. However, sensorimotor post-stroke dysfunction (e.g. decreased walking speed) could have influenced the results. Analogous to a previously used goal-directed locomotor paradigm, a seated, joystick-driven navigation experiment, minimizing locomotor demands, was employed in individuals with and without post-stroke USN (USN+ and USN-, respectively) and healthy controls (HC). Methods Participants (n = 15 per group) performed a seated, joystick-driven navigation and detection time task to targets 7 m away at 0°, ±15°/30° in actual (visually-guided), remembered (memory-guided) and shifting (visually-guided with representational updating component) conditions while immersed in a 3D virtual reality environment. Results Greater end-point mediolateral errors to left-sided targets (remembered and shifting conditions) and overall lengthier onsets in reorientation strategy (shifting condition) were found for USN+ vs. USN- and vs. HC (p < 0.05). USN+ individuals mostly overshot left targets (− 15°/− 30°). Greater delays in detection time for target locations across the visual spectrum (left, middle and right) were found in USN+ vs. USN- and HC groups (p < 0.05). Conclusion USN-related attentional-perceptual deficits alter navigation abilities in memory-guided and shifting conditions, independently of post-stroke locomotor deficits. Lateralized and non-lateralized deficits in object detection are found. The employed paradigm could be considered in the design and development of sensitive and functional assessment methods for neglect; thereby addressing the drawbacks of currently used traditional paper-and-pencil tools.