Mistreatment can adversely affect students’ academic performance and mental health. This study aimed to investigate the prevalence of mistreatment and its association with academic motivation among Thai medical students in clinical years. A cross-sectional study was conducted among medical students throughout Thailand using the Composite Questionnaire of Mistreatment and Academic Motivation Scale (AMS). Descriptive statistics, chi-square tests, correlations, and regression analyses were conducted. A p-value < 0.05 was considered statistically significant. The sample included 399 students, with mean age of 23 ± 1.29 years old, and 61.5% were female. A total of 81% of participants experienced mistreatment. Twenty-nine percents of students thought that mistreatment occurred because the maltreaters did not consider mistreatment as inappropriate or serious. Students who experienced mistreatment exhibited lower AMS (B = -9.16, 95% CI: -15.37 to -2.95, p-value = 0.004) than those who did not. Moreover, students who experienced academic mistreatment exhibited a higher amotivation score (B = 1.50, 95% CI: 0.16 to 2.85, p-value = 0.029) than those who did not. The prevalence of mistreatment was unexpectedly high, and it associated with students' motivation to study. Educational institutions should prioritize addressing this issue and developing strategies to provide assistance, including the enhancement of reporting systems.