Previous studies have revealed that patients with chronic fatigue syndrome and affective disorders (such as depression and anxiety disorders) exhibit a vigilant attentional bias toward negative emotional stimuli. However, it remains unclear whether the change in an attentional bias for negative emotional stimuli can be induced by mental fatigue in healthy individuals. To address this question, we examined healthy participants' (n = 27) performance in a visual probe task and emotional Stroop task before and after the mental-fatigue-inducing task. We demonstrated that acute mental fatigue induced by the long-lasting working memory task led to the alteration of cognitive processing of negative emotional information in the healthy volunteers.