Surgeons’ mental and physical workloads are major focuses of operating room (OR) ergonomics, and studies on this topic have generally focused on either mental workload or physical workload, ignoring the interaction between them. Previous studies have shown that physically demanding work may affect mental performance and may be accompanied by impaired mental processing and decreased performance. In this study, 14 participants were recruited to perform laparoscopic cholecystectomy (LC) procedures in a virtual simulator. Surface electromyography (sEMG) signals of the bilateral trapezius, bicipital, brachioradialis and flexor carpi ulnaris (FCU) muscles and eye-tracking signals were acquired during the experiment. The results showed that the least square means of muscle activity during the LC phases of surgery in an all-participants mixed effects model were 0.79, 0.81, and 0.98, respectively. The observed muscle activities in the different phases exhibited some similarity, while marked differences were found between the forearm bilateral muscles. Regarding mental workload, significant differences were observed in pupil dilation between the three phases of laparoscopic surgery. The mental and physical workloads of laparoscopic surgeons do not appear to be generally correlated, although a few significant negative correlations were found. This result further indicates that mental fatigue does markedly interfere with surgeons’ operating movements.