Muscle fatigue is a major constraint on physical performance and is driven by multiple factors such as excessive oxidative stress and ATP depletion. This study investigated the protective effects of licochalcone B (LicoB), a phenolic chalcone compound isolated from Glycyrrhiza glabra Linné, on oxidative stress-induced damage in C2C12 cells. Additionally, its anti-fatigue potential was evaluated in mice subjected to exhaustive swimming. In C2C12 myotubes, LicoB significantly reduced H2O2-induced oxidative damage and preserved myotube integrity by scavenging free radicals. Transcriptomic analysis of C2C12 cells showed that LicoB reversed stress-induced dysregulation of antioxidant and myogenic genes, indicating protective transcriptional reprogramming. An in vivo mouse model employing weight-loaded forced swimming further supported the beneficial effects of LicoB. Four weeks of oral administration of LicoB improved physical endurance, increased liver glycogen content, antioxidant enzyme activity in muscle, and blood free fatty acid level, while reducing circulating blood corticosterone and lactate levels, as well as lipid peroxidation in muscle tissues. Upregulation of TP53-induced regulator of glycolysis and apoptosis, sirtuin 1, and peroxisome proliferator-activated receptor-γ coactivator 1α in skeletal muscles suggested enhanced mitochondrial function and reduced oxidative damage. Additionally, transcriptomic analysis of skeletal muscle indicated that LicoB induced gene programs involved in myogenesis, neuromuscular junctions, and glucose metabolism. Collectively, these findings suggest that LicoB plays multi-targeted protective roles against excessive exercise-induced muscle fatigue by attenuating oxidative stress and modulating energy metabolism. Therefore, LicoB is a promising nutraceutical candidate for improving muscle endurance and recovery after strenuous physical activity.