The aseptic technique is essential for minimizing contamination in cell and tissue culture. However, biomedical science undergraduates in Universiti Putra Malaysia face challenges in acquiring hands-on learning experience on proper aseptic techniques due to inadequate facilities, which decreases learning interest and efficiency. To address this, an in-house virtual reality (VR) mobile application, AsepticTech VR, was developed to simulate a cell and tissue culture facility for practicing aseptic techniques. This study aims to investigate the efficacy of AsepticTech VR as a teaching and learning tool. Forty-one biomedical undergraduates were randomly assigned into test and control groups. Both groups attended a pre-test to evaluate the pre-intervention learning performances. Then, only test group participants were given access to AsepticTech VR, while both groups attended traditional lectures and practical sessions for a week. After one week, all participants took a post-test to evaluate their post-intervention learning performances. Additionally, test group participants also completed the modified Differential Emotions Scale (mDES) questionnaire to assess their emotions associated with using AsepticTech VR, and Web-Based Learning Tool (WBLT) questionnaire to assess their learning experience. The results showed that the test group participants had better learning performance improvements in the cognitive (p < 0.05) and psychomotor domain (p < 0.01) compared to the control group but did not differ in their affective domain. Moreover, the test group demonstrated stronger positive emotions compared to negative emotions (p < 0.001) and had positive learning experience with AsepticTech VR. This indicates that AsepticTech VR is an effective teaching and learning tool for cell and tissue culture aseptic techniques.