Inhibitory effects of ethanolic extracts from 10 Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. By a bioassay-guided fractionation procedure, NN-B-5 was identified from seeds of N. nucifera. NN-B-5 significantly blocked HSV-1 multiplication in HeLa cells without apparent cytotoxicity. To elucidate the point in HSV-1 replication where arrest occurred, a set of key regulatory events leading to the viral multiplication was examined, including HSV-1 DNA synthesis and viral immediate early gene expressions. Data from polymerase chain reaction and Southern blotting showed that there were impairments of HSV-1 DNA replication in HeLa cells treated with NN-B-5. Results indicated that the production and mRNA transcription of infected cell protein (ICP) 0 and ICP4 were decreased in NN-B-5 treated HeLa cells. Results of an electrophoretic mobility shift assay demonstrated that NN-B-5 interrupted the formation of alpha-trans-induction factor/C1/Oct-1/GARAT multiprotein/DNA complexes. The mechanisms of antiviral action of NN-B-5 seem to be mediated, at least in part, through inhibition of immediate early transcripts, such as ICP0 and ICP4 mRNA and then blocking of all downstream viral products accumulation and progeny HSV-1 production.