Herpes viruses (HSV) are among the most common viruses that infect humans. They cause blisters and inflammation of the oral and genital mucosa as well as severe symptoms such as blindness, hearing impairment and fatal encephalitis. Although acyclovir (ACV) and its derivatives are highly effective in treating recurrent lytic infections, the emergence of resistant strains remains a concern. In this study, the in vitro anti-herpetic activity of a hydroalcoholic extract obtained from the brown seaweed Sargassum fluitans, collected from strandings in Cuba, was evaluated. The hydroalcoholic extract was not cytotoxic in Vero cells (CC 50 >5000 µg.mL-1) and inhibited replication of HSV-1 (ACV-susceptible and-resistant) and HSV-2 (ACV-resistant) strains with EC 50 of 271.66, 403.62 and 14.99 µg.mL-1 respectively. In addition, first studies about mechanism of action were evaluated with ACV-sensitive HSV-1 model. The extract showed strong inhibition when pre-incubated with the virus and when the extract and virus were added simultaneously on the cells. The protection of Vero cells during the post-infection assay was very effective up to 1 h. A high resolution UPLC/MS-MS analysis detected 139 compounds, of which 44 were identified. In silico analysis was performed to determine the putative interaction sites of two flavones of interest identified in the extract with the viral glycoprotein D (gD). Molecular docking analysis revealed that takakin and tectorigenin mainly interact with the gD protein active center through hydrophobic interactions (Leu44, Pro50, Thr174, Lys169, Tyr208, Gln209) while hydrogen bonding interactions are less abundant (Ser43, Asp46, Lys169, Tyr208). This research represents a contribution to the valorization of S. fluitans as a source of anti-herpetic compounds in the context of recurrent and abundant seaweed strandings.