White spot syndrome virus (WSSV) is the most contagious pathogen causing huge economic losses in the shrimp industry worldwide. Several medicinal plants are known to have antiviral activity through the inhibition of viral diseases in fish and shellfish. Hence, there is a need to investigate the ability of natural remedies like plant sources to mitigate this devastating disease in crustaceans. This study was carried out to test the antiviral activity of ethanolic extract of Cynodon dactylon on WSSV in tiger shrimp Penaeus monodon juveniles with an average weight of 13.541 ± 2.927 g. Different doses of the extract (75, 100, and 150 mg/kg) were administrated in vivo through intramuscular injection. The antiviral activity was determined by observing survival rates, and WSSV infection was confirmed at the end of the experiment through polymerase chain reaction (PCR) identification. Before the in vivo experiment, presence of antiviral compounds ((+)-catechin, vanillic acid, syringic acid, (−)-epicatechin, p-coumaric acid, and quercetin bioactive compounds) in C. dactylon was confirmed through high-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis. The results obtained in this study show that a dose of 100 to 150 mg/kg body weight of 100 to 150 mg/kg body weight, ethanolic extract of C. dactylon ethanolic extract of C. dactylon prevented the infection of WSSV marked by 100% survival and absence of WSSV-specific band using nested diagnostic PCR, thus demonstrating the suitability of ethanolic extract of C. dactylon as a possible prophylactic for the prevention of WSSV infections in shrimp culture.