Azadirachta indica leaf is used by Indian population for the healing of various diseases including viral infection. Herein, we analyzed the antiherpetic (HSV-1) activity of two polysaccharides (P1 and P2) isolated from the leaf of A. indica and their chemically sulfated derivatives (P1S and P2S). The molecular weights of P1S and P2S are 41 and 11 kDa, respectively. Sulfate groups are located at positions C3 of the Araf and C6 of both Galp and Glcp residues of the most active polysaccharide (P1S). These compounds were not cytotoxic in HEp-2 cells, up to 1000 μg/mL. Both P1S and P2S exhibited antiviral activity when used simultaneously to HSV-1, with 50% inhibitory concentration/selectivity index, respectively, of 31.1 μg/mL/>51.4 and 80.5 μg/mL/>19.8. P1S showed better inhibitory effect (91.8%) compared to P1 (50%), P2 (71.1%) and P2S (70%) at 200 μg/mL. Synthesis of viral protein showed a dose-dependent response and the nucleic acid synthesis was inhibited up to 25 μg/mL, by P1 and P1S and up to 50 μg/mL, by P2 and P2S. The antiviral effect is probably due to the interference of polysaccharides at the early stages of HSV-1 replication, including adsorption. Further studies are under way to get insight into the mechanism of action of the substances.