Plantago major linn. and P. asiatica Linn. (Plantaginaceae) are commonly used as folk medicine in Taiwan for treating infectious diseases related to the respiratory, urinary and digestive tracts. In this study, we investigated the antiviral, cytotoxic and immunomodulatory activities of hot water extracts of these two species in vitro on a series of viruses, namely herpesviruses (HSV-1 and HSV-2), adenoviruses (ADV-3, ADV-8 and ADV-11), and on various human leukemia, lymphoma and carcinoma cells with XTT, BrdU and IFN-gamma kits. Results showed that hot water extract of P. asiatica possessed significant inhibitory activity on the proliferation of lymphoma (U937) and carcinoma (bladder, bone, cervix, kidney, lung and stomach) cells and on viral infection (HSV-2 and ADV-11). P. major and P. asiatica both exhibited dual effects of immunodulatory activity, enhancing lymphocyte proliferation and secretion of interferon-gamma at low concentrations (< 50 microg/ml), but inhibiting this effect at high concentration >> 50 microg/ml). The present study concludes that hot water extracts of P. major and P. asiatica possess abroad-spectrum of antileukemia, anticarcinoma and antiviral activities, as well as activities which modulate cell-mediated immunity. Further investigations to elucidate the active component(s) of P. asiatica and P. major and to evaluate their clinical application are warranted.