This study aimed to measure the spectral power differences in the brain rhythms among a group of hospital doctors before and after an overnight on-call duty. Thirty-two healthy doctors who performed regular on-call duty in a tertiary hospital in Sarawak, Malaysia were voluntarily recruited into this study. All participants were interviewed to collect relevant background information, followed by a self-administered questionnaire using Chalder Fatigue Scale and electroencephalogram test before and after an overnight on-call duty. The average overnight sleep duration during the on-call period was 2.2 hours (p<0.001, significantly shorter than usual sleep duration) among the participants. The mean (SD) Chalder Fatigue Scale score of the participants were 10.8 (5.3) before on-call and 18.4 (6.6) after on-call (p-value < 0.001). The theta rhythm showed significant increase in spectral power globally after an overnight on-call duty, especially when measured at eye closure. In contrast, the alpha and beta rhythms showed reduction in spectral power, significantly at temporal region, at eye closure, following an overnight on-call duty. These effects are more statistically significant when we derived the respective relative theta, alpha, and beta values. The finding of this study could be useful for development of electroencephalogram screening tool to detect mental fatigue.