Mosquitoes are medically important insect pests that act as vectors of various diseases when they feed on humans. Insect growth regulators (IGRs) could become an effective alternative to control mosquitoes because of their high specificity and relatively low toxicity to environment. Recently, we have developed high-throughput juvenile hormone antagonist (JHAN) screening system based on yeast-two hybrid assay. Actinomycetes have been reported to produce various bioactive compounds including insect growth regulators (IGRs). In this study, culture filtrates and mycelia extracts of 2,875 actinomycetes isolates screened for their IGR activities and mosquitocidal activities. Among 75 culture filtrates and 17 mycelia extracts with JHAN activities, 19 culture filtrate and 3 mycelia extracts showed high level of mosquitocidal activities against 3rd instar larvae of Aedes albopictus. These results suggested that secondary metabolites of actinomycetes could be used as environmentally benign mosquitocidal agents.