Human rhinoviruses (HRVs), members of the Picornaviridae family, are composed of over 100 different virus serotypes. Until now there is no recorded clinically effective antiviral chemotherapeutic agent for treatment of diseases caused by HRVs. Our previous study of raoulic acid tested against serotype human rhinoviruses showed anti-HRV2 (species A) and -3 (species B) activities. In this study, raoulic acid was found to possess broad-spectrum antiviral activity against six HRVs with a 50% inhibition concentration of less than 9.5 microg/mL through inhibition of the cellular absorption of the HRV particles. Furthermore, the effect of raoulic acid on resistance of HRV5 exhibited to pleconaril was more pronounced than the effect on HRV1b, -6, -14, -15, and -40. However, ribavirin did possess weak antiviral activity against HRVs. Collectively, the results demonstrate that raoulic acid is a novel therapeutic candidate for two different groups of human rhinovirus.