Abstract: The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species by using fungus-infested solid substrates as application material in this laboratory. We examined the effect of host-plant growth stage, dosage of inoculum, soil moisture condition, temperature, and damp soil duration on plant mortality and fresh weight inhibition of eclipta (Eclipta prostrata) caused by isolate SC64 in pot experiments under greenhouse conditions. Perfect control efficacy was recorded following application before 5 leaf stage, with over 100 g·m^-2 fungus-infested substrates (rice husk/bran substrate, 2:1, v/v), and exposure of weeds to 27-33 ℃, soil of 90% water holding capacity and at least 24 h damp soil duration. A simulation experiment of dry-seeded rice was carried out to examine the performance of S. rolfsii SC64 to broadleaf weeds under greenhouse conditions. Results showed that isolate SC64 (120 g·m^-2) caused 81% and 74% plant mortality, 83.8% and 82.5% fresh weight reduction for Rotala indica and Monochoria vaginalis, respectively. Plant mortality and fresh weight reduction for Cyperus difformis and Ammannia arenaria were relatively lower, which were approximately 50% and 60%-65%, respectively. It was indicated that this fungus could be a good candidate for further study and a promising biocontrol agent against broadleaf weeds under specific environmental conditions.