This study describes the in vitro antiviral activity of two species of oyster hemolymph (Crassostrea rhizophorae and C. gigas) collected on Southern Brazilian coastline. Either the acellular and cellular fractions were tested against herpes simplex type-1 (HSV-1), human adenovirus respiratory strain (AdV-5) and simian rotavirus (RV-SA11) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The evaluation of viral inhibition was performed using four different strategies: simultaneous, pre-infection and post-infection treatments and virucidal assays. The cellular fraction from Crassostrea rhizophorae showed the most prominent inhibition of HSV-1 and AdV-5 replication, particularly in post-infection treatment assay. No inhibition of rotavirus replication was observed. Both oyster hemolymph fractions showed virucidal activity against all tested virus in non-cytotoxic concentrations.