Mental fatigue is inevitable in the workplace. Since mental fatigue can lead to decreased efficiency and critical accidents, it is important to manage mental fatigue from the viewpoint of accident prevention. An experiment was performed to evaluate mental fatigue using the formant frequency analysis of human voices. The experimental task was to mentally add or subtract two one-digit numbers. After completing the tasks with four different levels of mental fatigue, subjects were asked to read Korean vowels and their voices were recorded. Five vowel sounds of “아”, “어”, “오”, “우”, and “이” from the voice recorded were then used to extract formant 1 frequency. Results of separate ANOVAs showed significant main effects of mental fatigue on formant 1 frequencies of all five vowels concerned. However, post-hoc comparisons revealed that formant 1 frequencies of “아” and “어” were most sensitive to mental fatigue level employed in this experiment. Formant 1 frequencies of “아” and “어” significantly decrease as the mental fatigue accumulates. The formant frequency extracted from human voice would be potentially applicable for detecting mental fatigue induced during industrial tasks.