The aim of present study was to evaluate the symmetry of masticatory muscles' activity at various clenching levels in the intercuspal position in patients with functional disorders and in healthy subjects. The purpose was also to determine the effect of full-arch maxillary stabilization splint on the asymmetry of masticatory muscle activity in patients with temporomandibular dysfunction. In this study 6 TMD patients and 12 healthy subjects were investigated. Surface EMG recordings were obtained from left and right anterior temporal, left and right masseter and from the sub-mandibular group in the region of the anterior belly of the digastric muscle on the left and right side during clenching with the maximum 100% voluntary contraction (MVC) as well as during clenching at 50% and 25% of the maximum activity in the position of maximal intercuspation of teeth. In order to quantify asymmetrical masticatory muscle activity, the asymmetry index (AI) was calculated for each subject and for each muscle from the average anterior temporal, masseter and digastric potentials recorded during each test (100% MVC, 50% MVC and 25% MVC). In the group of patients EMG recordings were repeated during and after the splint therapy. The asymmetries of masticatory muscle activity was present in both groups, but in the group of TMD patients the asymmetry indices for anterior temporal muscle at 100% MVC (p = 0.049) and 50% MVC (p = 0.031) were significantly higher. Results have shown that the use of splint suppressed the asymmetry of all muscles, as during the splint therapy the asymmetry indices were lowered. After the therapy, the level of temporal muscle symmetry during submaximal clenching in the intercuspal position increased significantly (p = 0.046). This investigation points out that electromyography may be a valuable method of documenting that asymmetric activity of masticatory muscles improves after occlusal splint therapy in patients with TMD.