This study was conducted to examine the effects and the mode of action of a natural herbicidal substance, Streptomyces scopuliridis metabolites (SSM), which consists of herbicidin A and herbicidin B, derived from Streptomyces scopuliridis. Necrosis of Echinochloa oryzoides, Digitaria ciliaris, Abutilon theophrasti and Amaranthus retroflexus occurred within 3 days of SSM application at the concentration of 4,000 ppm. Symptoms of herbicidal action of SSM started to show soon following application, and appeared earlier than those caused by glyphosate but later than those caused by paraquat. Overall, herbicidal action of SSM appeared with a time interval similar to glufosinate ammonium following foliar application. Chlorophyll fluorescence level (Fv/Fm) was not affected by SSM application suggesting that SSM application did not inhibit photosynthesis of the treated plants. The amount of electrolyte leakage caused by SSM increased steadily with time, and was proportional to the concentration of SSM. Electrolyte leakage induced by paraquat occurred more rapidly than by SSM. No signs of translocation of SSM in a downward direction through the phloem was observed in either D. ciliaris or A. theophrasti. Based on the results from this study, the mode of action of SSM involves the rapid disruption of cell membrane caused by an unknown mechanism other than inhibition of photosynthesis. In the future, SSM would be a good candidate for a contact herbicide; however, a better understanding of its mode of action has to precede attempts for commercial development and practical application.