The antiviral effect against RVA in cell culture was evaluated by using an aqueous extract of Patallus mollis sea cucumber, applying the titration methodology. This technique is used to measures the ability of the extract dilutions to inhibit the cytopathic effect (CPE) of the virus, expressed as percentage of inhibition (IP). The mean extract cytotoxic concentration (CC50) used in the antiviral assay was 27,042.10 μg/mL and the PI of the antiviral activity extract was greater than 99.9% for each concentration. To determine the viral action mode, the cells were previously treated with the extracts in different stages during the viral infection cycle. The result analysis suggests that the extract inhibits 99% of the virus during the absorption and viral inactivation phase. These results show the P. mollis extract has a remarkable antiviral effect against the RVA in cell culture. So that, it is crucial to investigate its action mechanisms.