The aim of the present study was to analyze novel functional indices of masticatory muscle activity and compare them to existing and commonly used indices in patients with temporomandibular disorders (TMDs) and healthy adults. Based on the Research Diagnostic Criteria for Temporomandibular Disorders, 78 adult women qualified for the study. Subjects were divided into two groups: diagnosed TMDs (n = 36; mean age: 23.4 ± 2.6 years) and healthy adults (n = 42; mean age: 22.4 ± 2.3 years). Measurements of the bioelectric activity of the temporalis anterior (TA), superficial masseter (MM), and anterior bellies of the digastric muscle (DA) were carried out using the BioEMG III ™. Functional Clenching (FCI) and Functional Opening (FOI) indices were obtained as the ratio of the difference between the mean muscle root mean square (RMS) potentials during functional activity, including clenching (CL) and opening (MMO), and mean muscle resting (REST) potentials. Next, based on FCI and FOI indices, the Functional Clenching Activity Index (FCAI), Functional Clenching Symmetry Index (FCSI), and Functional Opening Symmetry Index (FOSI) were obtained. The statistical analysis showed significant differences in activity index left-sided (AcIL) and Activity index both-sided (AcItot) between TMDs and healthy women during rest measurements. The significant differences between both groups were noted in terms of all Functional Clenching Indices except Functional Clenching Index for MM right-sided (FCIMM-R). In all analyzed FCI indices, the control group showed higher values compared to the TMDs. Moreover, a significant difference between TMDs and controls was observed within Functional Clenching Activity Index left-sided (FCAIL) (14.56 vs. −0.45, p = 0.01). Both functional indices, and asymmetry (AsI) and activity (AcI) indices seem to be reliable in assessing symmetry and activity within masticatory muscles. Further studies should be performed to verify the effectiveness and suitability of the assessment of masticatory muscles using functional indices.