Rhamnan sulfate (RS), a natural sulfated polysaccharide isolated from Monostromo latissimum, showed potent inhibitory effects on the virus replication of herpes simplex virus type 1 (HSV-1), human cytomegalovirus (HCMV), and human immunodeficiency virus type 1 (HIV-1) in vitro. The antiviral action of RS was not only due to the inhibition of virus adsorption, but also might involve the later steps of viral replication in host cells on the basis of the results of time-of-addition experiments. Furthermore, RS and 3′-azido-3′-deoxythymidine (AZT) were synergistic in their anti-HIV-1 activities. These data indicate that RS is a potent antiviral substance against HSV-1, HCMV, and HIV-1.