Sulfated polysaccharides are good candidates for drug discovery in the treatment of herpetic infections. Agaricus brasiliensis (syn A. subrufescens, A. blazei) is a Basidiomycete fungus native to the Atlantic forest region of Southeastern Brazil. Herein we report the chemical modification of a polysaccharide extracted from A. brasiliensis mycelia to obtain its sulfated derivative (MI-S), which presented a promising inhibitory activity against HSV-1 [KOS and 29R (acyclovir-resistant) strains] and HSV-2 strain 333, with selectivity indices (SI = CC50/IC50) higher than 439, 208, and 562, respectively. The mechanisms underlying this inhibitory activity were scrutinized by plaque assay with different methodological strategies. MI-S had no virucidal effects, but inhibited HSV-1 and HSV-2 attachment, penetration, and cell-to-cell spread, as well as reducing the expression of HSV-1 ICP27, UL42, gB, and gD proteins. MI-S also presented synergistic antiviral effect with acyclovir. These results suggest that MI-S presents multiple modes of anti-HSV action.