Objective: The goal of this study is to detect the efficacy of Pilates mat exercise and conventional therapy on cervical flexor and extensor myoelectric amplitude in subjects with chronic mechanical neck pain. Methods: This trial included 91 participants from both genders (42 males and 49 females) who were diagnosed by an orthopedist with chronic mechanical neck pain and referred to the physiotherapy clinic. The participants were randomly assigned to two groups: Group A, which is an experimental group, included those receiving Pilates mat exercise in conjunction with conventional therapy, and Group B, which is a control group, included those receiving only conventional therapy. The intervention was delivered to both groups three times a week for 12 weeks. In this study, the variables pain, function, and muscle activity were measured using the visual analog scale, Arabic neck disability index, and Electromyography respectively. All variables were assessed before and after the 12 weeks. Results: To detect the impact of treatment and time on all measured variables, MANOVA was used and showed a statistically significant effect (p < 0.05) in both groups in treatment and time with favor to Pilate's group. Conclusion: Pilates mat exercise combined with conventional therapy and conventional therapy alone are effective techniques for refining neck pain, function, and muscle amplitude in subjects with chronic mechanical neck pain, with further advantages shown in the experimental group that received the Pilates mat exercise treatment.