The presented study aimed to analyze and compare the electromyographic patterns of masticatory muscles in subjects with active myofascial trigger points (MTrPs) within upper trapezius, patients with temporomandibular disorders (TMDs) and healthy adults. Based on the diagnostic criteria of MTrPs according to Travell & Simons and the Research Diagnostic Criteria for Temporomandibular Disorders, 167 people were qualified for the study. Subjects were divided into 3 groups: with active MTrPs in the upper trapezius, with diagnosed temporomandibular disorders (TMDs) and healthy adults. Measurements of the bioelectric activity of the temporalis anterior (TA) and masseter muscle (MM) were carried out using the BioEMG III ™. Based on statistical analysis, significantly lower values of TA resting activity were observed among controls in comparison to MTrPs (1.49 μV vs. 2.81 μV, p = 0.00) and TMDs (1.49 μV vs. 2.97 μV, p = 0.01). The POC index values at rest differed significantly between MTrPs and TMDs (86.61% vs. 105%, p = 0.04). Controls presented different electromyographic patterns within AcI in comparison to both MTrPs (4.90 vs. −15.51, p = 0.00) and TMDs (4.90 vs. −16.49, p = 0.00). During clenching, the difference between MTrPs and TMDs was observed within MVC TA (91.82% vs. 116.98%, p = 0.02). TMDs showed differences within AcI in comparison to both MTrPs group (−42.52 vs. 20.42, p = 0.01) and controls (−42.52 vs. 3.07, p = 0.00). During maximum mouth opening, differences between MTrPs and TMDs were observed within the bioelectric activity of masseter muscle (16.45 μV vs. 10.73 μV, p = 0.01), AsI MM (0.67 vs. 11.12, p = 0.04) and AcI (13.04 vs. −3.89, p = 0.01). Both the presence of MTrPs in the upper trapezius and TMDs are related to changes in electromyographic patterns of masticatory muscles.