Objectives: This study aims to explore effects of ultrasound-assisted injection of the botulinum neurotoxin-A (BoNT-A) on muscle thickness (MT) in patients with masseter hypertrophy. Patients and methods: Between December 2018 and December 2019, a total of 20 patients (3 males, 17 females; mean age: 28±6.9 years; range, 18 to 42 years) with bruxism who underwent BoNT-A injections were reviewed retrospectively. The patients were treated using individualized injection dosages and sites. Assessment methods included length between the tragus and the angle of the mouth, maximal bite force, and MT of the masseter muscle. Follow-up data were collected from the hospital electronic database. Results: There were 26 masseter muscles with masseter hypertrophy in 20 patients. There was a significant difference in ultrasonographic measurements of the relaxed and contracted masseter muscles between the baseline and two weeks, one month and three months after the treatment (p<0.05). In the ultrasonographic measurement of the relaxed masseter muscle, post-treatment third month values significantly differed from the second week values. The differences in the measurement of the line between the tragus and the angle of the mouth between the baseline and two weeks, one month and three months after the treatment were statistically significant (p<0.05). In the maximal bite force measurements, no significant difference was observed between the baseline and post-intervention measurements (p>0.05). Conclusion: The MT decreases after a single dose of BoNT-A injection in patients with masseter muscle hypertrophy and ultrasonography is a convenient imaging modality for BoNT-A injection to the masseter.