The aim of our study was to assess university students' sleep quality and fatigue before and during the academic exam period and identify potential associated factors. A Web-based survey was completed by 940 students of 20 different Tertiary Institutions including demographics, sleep habits, exercise, caffeine, tobacco, alcohol use, subjective sleep quality (Pittsburgh Sleep Quality Index-PSQI), and fatigue (Fatigue severity scale-FSS) at the beginning of the semester and during the examination period. During the exam period, PSQI (8.9 vs. 6.1, p < 0.001) and FSS scores (36.9 vs. 32.7, p < 0.001) were significantly elevated compared to the pre-exam period. An increase in the PSQI score was associated with age (β = 0.111, p = 0.011), presence of chronic disease (β = 0.914, p = 0.006), and depressive symptoms (β = 0.459, p = 0.001). An increase in the FSS score was associated with female gender (β = 1.658, p < 0.001), age, (β = 0.198, p = 0.010), increase in smoking (β = 1.7, p = 0.029), coffee/energy drinks consumption (β = 1.988, p < 0.001), decreased levels of physical exercise (β = 1.660, p < 0.001), and depressive symptoms (β = 2.526, p < 0.001). In conclusion, our findings indicate that exam periods have a negative impact on the sleep quality and fatigue levels of university students. Potential factors were identified that could contribute to the formulation of strategies for improved sleep quality and wellness.