Spatial visualization ability (SVA) has been identified as a potential key factor for academic achievement and student retention in Science, Technology, Engineering, and Mathematics (STEM) in higher education, especially for engineering and related disciplines. Prior studies have shown that training using virtual reality (VR) has the potential to enhance learning through the use of more realistic and/or immersive experiences. The aim of this study was to investigate the effect of VR-based training using spatial visualization tasks on participant performance and mental workload using behavioral (i.e., time spent) and functional near infrared spectroscopy (fNIRS) brain-imaging-technology-derived measures. Data were collected from 10 first-year biomedical engineering students, who engaged with a custom-designed spatial visualization gaming application over a six-week training protocol consisting of tasks and procedures that varied in task load and spatial characteristics. Findings revealed significant small (Cohen’sd: 0.10) to large (Cohen’sd: 2.40) effects of task load and changes in the spatial characteristics of the task, such as orientation or position changes, on time spent and oxygenated hemoglobin (HbO) measures from all the prefrontal cortex (PFC) areas. Transfer had a large (d= 1.37) significant effect on time spent and HbO measures from right anterior medial PFC (AMPFC); while training had a moderate (d= 0.48) significant effect on time spent and HbR measures from left AMPFC. The findings from this study have important implications for VR training, research, and instructional design focusing on enhancing the learning, retention, and transfer of spatial skills within and across various VR-based training scenarios.