Background: Non-contact anterior cruciate ligament (ACL) injuries are prevalent among basketball players, particularly during abrupt stop-jump actions. Most existing studies have focused primarily on the impact of physical fatigue on athletic performance, often overlooking the critical role of mental fatigue (MF). The MF experienced in high-intensity game scenarios may further increase the risk of non-contact ACL injuries by altering lower limb biomechanical characteristics; however, the underlying mechanisms remain unclear. This study aimed to investigate the effects of mental fatigue on lower limb biomechanics during stop-jump tasks in basketball players and evaluate its potential contribution to ACL injury risk factors. Methods: A total of 38 basketball players were recruited via a self-controlled study design. MF was induced through a 45-minute Stroop task, with fatigue levels assessed via the visual analogue scale (VAS). Infrared motion capture systems, force platforms, and surface electromyography (EMG) devices were used to measure changes in kinematic, kinetic, and surface EMG data before and after MF intervention. The Wilcoxon signed-rank test was applied to analyse the effects of MF on lower limb biomechanical characteristics. Results: Following the induction of MF, significant decreases were observed in the knee flexion angle (z = -2.211, P = 0.02), internal rotation angle (z = -2.228, P = 0.02), internal rotation moment (z = -2.178, P = 0.02), and median frequency (MDF) of the rectus femoris (z = -3.009, P < 0.01), biceps femoris (z = -3.285, P < 0.01), tibialis anterior (z = -3.053, P < 0.01), and gastrocnemius (z = -2.110, P = 0.03). Conversely, significant increases were noted in the peak vertical ground reaction force (z = -2.015, P = 0.04) and knee extension moment (z = -2.162, P = 0.03). Conclusions: MF negatively impacts the biomechanics of the lower extremities during abrupt stop jumps in basketball players, potentially increasing the risk of non-contact ACL injury.