Virtual microscopy (VM) has been utilized to improve students' learning experience in microscope laboratory sessions, but minimal attention has been given to determining how to use VM more effectively. The study examined the influence of VM on academic performance and teacher and student perceptions and compared laboratory test scores before and after VM incorporation. A total of 662 third-year students studying histology and 651 fourth-year students studying pathology were divided into two groups. The light microscopy (LM) group used a light microscope in 2014 and 2015, while the LM + VM group used the VM platform and a light microscope in 2016 and 2017. Four factors positively predict laboratory scores (R square, 0.323; P < 0.001): (1) the pathology course and test-enhanced learning; (2) the VM platform and experience; (3) medical students and lecture scores; and (4) female students. The LM + VM group exhibited less score variability on laboratory examinations relative to their mean than the LM group. The LM + VM group was also associated with fewer failing grades (F grade; odds ratio, 0.336; P < 0.001) and higher scores (A grade; odds ratio, 2.084; P < 0.001) after controlling for sex, school, course, and lecture grades. The positive effect of the VM platform on laboratory test grades was associated with prior experience using the VM platform and was synergistic with more interim tests. Both teachers and students agreed that the VM platform enhanced laboratory learning. The incorporation of the VM platform in the context of test-enhanced learning may help more students to master microscopic laboratory content.