This study evaluated the antiviral activity of the sulfated polysaccharides (SP) from three species of seaweeds, two agarans (Acanthophora muscoides and Gracila riabirdiae) and one carrageenan (Solieria filiformis) and the relationship of this activity with the position of the sulfate groups in the structure, the sulfate content and the molecular weight. Total sulfated polysaccharides (TSP) were extracted by enzymatic digestion and fractioned on DEAE-cellulose column. The fractions with the highest yield from each species were further studied (SP-Am; SP-Gb and SP-Sf). Fourier transform infrared spectroscopy (FT-IR) demonstrated specific signs of sulfate on three species. In addition, SP-Am presented the lowest molecular weight (<100 kDa) by gel permeation chromatography. The cell viability of SP was determined by the change of the Vero cell morphology. The degree of antiviral activity was expressed as percent inhibition of the herpesvirus. The selectivity index and IC50 were also performed. SP-Gb (1000 μg/ml), SP-Am (250 μg/ml) and SP-Sf (500 μg/ml) showed no toxicity on Vero cells and had an antiviral effect against HSV-1 and HSV-2, when compared with the control group. The antiviral effect was mainly exerted by SPAm, which presented the higher sulfate content and the lower molecular weight compared to SP-Gb and SP-Sf.