This study assessed the safety and efficacy of three different doses of BoNT-A for persistent myofascial pain (MFP). One hundred female subjects were randomly assigned into five groups (n = 20): oral appliance (OA), saline solution (SS) and three BoNT-A groups with different doses. Pain intensity and pressure pain threshold were evaluated up to 24 weeks after treatment. Adverse effects related to muscle contraction, masticatory performance, muscle thickness and mandibular bone volume were also assessed. Changes over time were compared within and between groups. The “nparLD” package and Wilcoxon signed-rank test were used to analyze the data. BoNT-A reduced pain intensity (p p p p p p < 0.05) were found as dose-related adverse effects of BoNT-A. Regardless of the dose, BoNT-A was as effective as OA on MFP. Notwithstanding, due to BoNT-A dose-related adverse effects, we suggest the use of low doses of BoNT-A in MFP patients that do not benefit from conservative treatments.