Da Vinci three-dimensional (3D) system has been increasingly used in customary surgical settings, gaining fundamental relevance for abdominal, urological, and gynecological laparoscopic surgery. The aim of this research is to evaluate the degree of discomfort and potential changes in the binocular vision and ocular motility of surgical operators, who employ 3D vision systems during Da Vinci robotic surgery. Twenty-four surgeons were enrolled in the study, including twelve who typically use the 3D Da Vinci system and twelve who routinely employ 2D system. Routine general ophthalmological and orthoptic examinations were conducted at baseline (T0), the day before surgery, and 30 min after the 3D or 2D surgery (T1). In addition, surgeons were interviewed using a questionnaire of 18 symptoms, with each item containing three questions regarding the frequency, severity, and bothersomeness of the symptoms, in order to evaluate the degree of discomfort. Mean age at evaluation was 45.28 ± 8.71 years (range 33–63 years). Cover test, uncover test, and fusional amplitude showed no statistically significant difference. After surgery, no statistical difference was observed in the Da Vinci group on the TNO stereotest (p > 0.9999). However, the difference in the 2D group resulted statistically significant (p = 0.0156). Comparing participants (p 0.0001) and time (T0–T1; p = 0.0137), the difference between the two groups was statistically significant. Surgeons using 2D systems reported more discomfort than those using 3D systems. The absence of short-term consequences following surgery with the Da Vinci 3D system is a promising conclusion, considering the numerous advantages of this technology. Nonetheless, multicenter investigations and more studies are required to verify and interpret our findings.