This cross-sectional study aimed to evaluate the prevalence of dry eye disease (DED) symptoms, their associated factors, and the impact on quality of life (QOL) and mental health among medical students during the COVID-19 pandemic using online surveys. Participants completed questionnaires covering demography, medical history, and personal data. The Thai version of the Dry Eye-related Quality-of-Life Score (DEQS-Th) was used for DED screening. The QOL and mental health challenges (support, coping, stress, and depression) were evaluated by the EuroQoL-5 dimensions-5 levels (EQ-5D-5L) and Thymometer questionnaires. A total of 449 participants were analyzed, with a mean age of 21.8 years and 61.5% female. The prevalence of DED symptoms was 60.4% (95%CI 55.7–64.8). The DEQS-Th score, the EQ-5D-5L score, and all aspects of mental health challenges were significantly worse in DED participants compared to non-DED participants. Associated factors for DED symptoms included preexisting DED (
p
= 0.001), refractive errors (
p
= 0.007), allergic conjunctivitis (
p
= 0.001), artificial tears use (
p
< 0.001), and decreased EQ-5D-5L score (
p
< 0.001). This study highlighted the high prevalence of DED symptoms among medical students during the pandemic and its negative impact on QoL and mental health. Notably, female gender, contact lens wear, screen time, and mask-wearing duration were not associated with DED symptoms.