Microsaccades, small saccadic eye movements occurring during fixation, have been suggested to be modulated by various sensory, cognitive, and affective processes relating to arousal. Although the modulation of fatigue-related arousal on microsaccade behavior has previously been characterized, the influence of other aspects of arousal, such as emotional arousal, is less understood. Moreover, microsaccades are modulated by cognitive processes (e.g., voluntary saccade preparation) that could also be linked to arousal. To investigate the influence of emotional arousal, saccade preparation, and global luminance levels on microsaccade behavior, emotional auditory stimuli were presented prior to the onset of a fixation cue whose color indicated to look either at the peripheral stimulus (pro-saccade) or in the opposite direction of the stimulus (anti-saccade). Microsaccade behavior was found to be significantly modulated by saccade preparation and global luminance level, but not emotional arousal. In the pro- and anti-saccade task, microsaccade rate was lower during anti-saccade preparation as compared to pro-saccade preparation, though microsaccade dynamics were comparable during both trial types. Our results reveal a differential role of arousal linked to emotion, fatigue, saccade preparation, and global luminance level on microsaccade behavior.