Substituted (E)-3-styryl-4H-chromen-4-ones 1a-d, 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones 2a-d, (E)-3-styryl-2H-chromenes 3a-d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes 4a-d were designed and synthesized to improve the anti-picornavirus activity of previously tested analogues. The new compounds were evaluated in vitro against human rhinovirus (HRV) serotypes 1B and 14 and enterovirus (EV) 71. All the compounds interfered with the replication of picornaviruses, although considerable differences were observed in the sensitivity of viruses to each compound. Generally, both HRVs were more susceptible than EV71 and their sensitivity was dependent upon the linker chain length as well as upon the oxidation state of the heterocyclic ring. (E)-3-Styryl-2H-chromene (3a) emerged as the most effective inhibitor of both HRVs showing IC(50) values of 0.20 microM and 1.38 microM towards serotype 1B and 14, respectively. The potent activity was also coupled with low cytotoxicity resulting in high therapeutic indexes (250 and 36, respectively). Mechanism of action studies indicated that 3a, like structurally related compounds, behaves as a capsid binder interfering with the early stages of rhinovirus infection, probably at the adsorption and/or uncoating level.