Searching for antiviral antibiotics, we found acetone extract of two strains of soil fungi exerted remarkable antiviral activity against Newcastle disease virus (NDV), vaccinia and herpes simplex viruses in agar diffusion-plaque inhibition method, and isolated the active principles in crystalline form, which were identified as xanthocillin X mono- and dimethylether, and methoxy-xanthocillin X dimethylether (XME, XDE and XTE, respectively). The three antibiotics had low cytotoxicity against primary culture of chick embryo fibroblast cells (CEF) at higher concentrations than that required for suppression of occurrence of cytopathic effect (CPE) following viral growth. Their antiviral activity was studied employing NDV and primary culture of CEF virus-cell system. Effects of XME, XDE and XTE on multiplication of NDV and on proliferation of CEF were examined. At low multiplicity, XME, XDE and XTE suppressed CPE at low concentrations. At high multiplicity, XME (10 mcg/ml) completely inhibited occurrence of CPE and syntheses of HA and infective virus, and at lower concentrations there was no difference in final yields of HA at 23 hours post infection, but a concentration dependency was found in synthesis of HA and suppression of CPE. XDE and XTE did not inhibit HA synthesis completely at high concentration, but delayed the onset of HA production and partially arrested the occurrence of CPE post infection. All three antibiotics had low cytotoxicity against CEF, and no effect on free NDV particle and on viral adsorption onto CEF. The present paper reports the effect on cell growth, on free virus particle and adsorption of NDV onto CEF and also the antiviral activity determined by tube assay.