Background: The impact of gender and hand dominance on operative performance may be a subject of prejudice among surgeons, reportedly leading to discrimination and lack of professional promotion. However, very little objective evidence is available yet on the matter. This study was conducted to identify factors that influence surgeons’ performance, as measured by a virtual reality computer simulator for laparoscopic surgery. Methods: This study included 25 surgical residents who had limited experience with laparoscopic surgery, having performed fewer than 10 laparoscopic cholecystectomies. The participants were registered according to their gender, hand dominance, and experience with computer games. All of the participants performed 10 repetitions of the six tasks on the Minimally Invasive Surgical Trainer—Virtual Reality (MIST-VR) within 1 month. Assessment of laparoscopic skills was based on three parameters measured by the simulator: time, errors, and economy of hand movement. Results: Differences in performance existed between the compared groups. Men completed the tasks in less time than women (p = 0.01, Mann–Whitney test), but there was no statistical difference between the genders in the number of errors and unnecessary movements. Individuals with right hand dominance performed fewer unnecessary movements (p = 0.045, Mann–Whitney test), and there was a trend toward better results in terms of time and errors among the residence with right hand dominance than among those with left dominance. Users of computer games made fewer errors than nonusers (p = 0.035, Mann–Whitney test). Conclusions: The study provides objective evidence of a difference in laparoscopic skills between surgeons differing gender, hand dominance, and computer experience. These results may influence the future development of training program for laparoscopic surgery. They also pose a challenge to individuals responsible for the selection and training of the residents.