Background. Anterior cruciate ligament (ACL) injuries represent a significant issue in competitive handball, frequently resulting in prolonged absence and diminished athletic performance. Given the sport’s high physical demands, particularly at the collegiate level, monitoring training load and fatigue is essential to mitigating injury risk. Objectives. This study aimed to examine the relationship between training load, markers of fatigue, and ACL injury risk in male collegiate handball players during two competition seasons. Materials and methods. A prospective cohort study was conducted with 93 male athletes (mean age: 20.8 ± 1.6 years) from 13 collegiate handball teams across two seasons (AY 2022-2024). Training load was tracked using weekly hours, session-RPE, and match exposure. The assessment of fatigue was performed through daily wellness ratings and countermovement jump (CMJ) height decline. ACL injury risk was calculated from a composite score including LESS assessments, limb asymmetry, and fatigue-related performance decline. Logistic regression was used to identify predictors of ACL injury, with positional differences examined via ANOVA. Results. Fatigue score was the only statistically significant predictor of ACL injury (p = 0.041). Players reporting higher fatigue levels and categorized within the high training load group exhibited the greatest ACL risk. Right and Centre Backs experienced greater training demands and fatigue accumulation compared to Left Backs. Conclusions. The findings indicate that persistent fatigue, more than training volume alone, significantly predicts ACL injury risk in collegiate handball players. Integrating regular fatigue monitoring and individualized load management into training programs may reduce injury rates and improve athlete longevity.