The effect of small anisometropia on visual fatigue when using virtual reality (VR) devices was investigated. Participants (n = 34) visited three times. In the first visit, VR exposure (10 minutes) was conducted with the full correction of the refractive error of both eyes. Experimental anisometropia was induced by adding a +1.0 diopter spherical lens either on the dominant eyes in the second visit or on the non-dominant eyes in the third visit. At each visit, the participants played a predetermined video game using a head-mounted display VR for 10 minutes. Visual fatigue was assessed before and after playing VR game using the Virtual Reality Symptom Questionnaire (VRSQ) and high-frequency component of accommodative microfluctuation. Results showed that watching VR induced significant increase of VRSQ score, significant decrease in the maximum accommodation power and objective increase in visual fatigue. Experimental anisometropia induction either on the dominant or non-dominant eyes did not aggravate visual fatigue. Mild differences in refractive error (up to 1.0 diopter) between both eyes do not significantly increase ocular fatigue by viewing virtual reality device (10 minutes). The impact of small anisometropia may be limited in developing a virtual reality device.