A chromene derivative (1) obtained from a brown alga, Sargassum micracanthum, has been proved to be a potent inhibitor of human cytomegalovirus (HCMV). In the present study, we evaluated its mode of action by various experimental assays. Time-of-addition experiments revealed that 1 was active if applied to cells before viral DNA synthesis, indicating that it inhibited early events of virus replication including virus adsorption and penetration, and a step immediately after viral internalization. Virus attachment and penetration studies suggested that one of the targets for anti-HCMV action of 1 was virus adsorption to cells and to a lesser extent, virus internalization was delayed in the presence of the compound. Pretreatment of virus particles with 1 showed that the compound exerted dose-dependent virucidal action. The chromene derivative and ganciclovir (GCV), an anti-HCMV drug, were synergistic inhibitors when used in combination. The synergistic effect could be explained by inhibition of different steps in HCMV replication cycle produced by 1 and GCV.