Objective: To investigate the action of osteopathic manipulative treatment on the muscular activity of the stomatognathic apparatus by using surface electromyography (sEMG). Material and Methods: Surface electromyography (sEMG) was performed on the masseter and anterior temporalis muscles of 120 subjects (73 F; 47 M), both at time T0 and T2. The sample was divided into three randomized groups of 40 subjects each: control, placebo, and osteopathic manipulative treatment (OMT). In the T1 interval between the two evaluations, the control group was not treated, the placebo group underwent a placebo treatment, and the OMT group underwent manipulative treatment. The mean value of each measurement and its coefficient of variation, between time T0 and T2, were calculated for both the intragroup (OMT, placebo, control) and the intergroup (OMT-placebo, OMT-control). Outcomes: In 40% of the subjects, statistically significant improvements were highlighted in the OMT. Whereas, the statistically significant results of the placebo and control groups were 7.5% and 17.5%, respectively, of which more than 75% moved away from the physiological range, showing a worsening of the muscular activity. This analysis showed statistically significant variations (p ≤ 0.05) in the OMT group compared to the placebo and the control groups. Conclusions: OMT determines variations of the activity of masticatory muscles.