An evaluation of thirty-four bufadienolides and two related cardenolides against a series of rhinoviruses in vitro has been completed. Most of the bufadienolides were found to display some inhibitory activity. Scillarenin and 3-O-[N-(tert-butoxycarbonyl)hydrazido]succinylbufalin were found to be the most active with chemotherapeutic indices of 32 and 16, respectively. In general, the 14β-hydroxy-bufadienolides showed the strongest antiviral activity, and were found more toxic than the corresponding 14β, 15β-epoxy-bufadienolides. Introduction of a 16β-hydroxy or 16β-acetoxy substituent into the 14β, 15β-epoxybufadienolides enhanced their antiviral activity. Substituents at the 3β-, 5β-, and 19-positions appeared to affect only the level of toxicity.