The present study studied the effects of fish gelatin (FG) incorporated with grape seed extract (GSE) through vacuum impregnation (VI) on refrigerated tilapia (Oreochromis niloticus) fillets over 12 days. The VI of FG-GSE significantly improved the quality of the fish by decreasing drip loss, texture changes, and microbial survival. It also delayed protein oxidation by inhibiting the formation of disulphide bonds and carbonyl groups, and maintaining a higher sulfhydryl content and Ca2+-ATPase activity. Regarding myofibril degradation, FG-GSE maintained their secondary structure by increasing the ratio of α-helices and β-sheets (70.88–75.51%). Atomic force microscopy further revealed that the FG-GSE coating preserved the myofibril nanostructure by maintaining their length, width, and height. Overall, the synergistic effects of VI with 3% FG and 0.9% GSE suggested a promising approach for fillet preservation.