The invention provides an agricultural fungicide WYLZ-1349 taking azalomycin F3, azalomycin F4 and a nyphimycin composition as effective constituents, as well as preparation method and application thereof. The preparation method of the agricultural fungicide WYLZ-1349 comprises the following steps of: firstly culturing Streptomyces malaysiensis ECO 00002 with the preservation number of CGMCCNo. 1680; then separately extracting a fermentation culture; breaking the wall of mycelium with acetone or methanol and mixing with a fermentation supernate; and absorbing by macroporous absorption resin for separation; eluting by the acetone or the methanol; concentrating or further purifying an eluent so as to obtain the agricultural fungicide WYLZ-1349 or the azalomycin F3, the azalomycin F4 and nyphimycin; and then adding a carrier or an auxiliary agent so as to obtain a finished product. Test results show that the agricultural fungicide WYLZ-1349 has good effect especially in preventing and controlling rice blast, tobacco brown spot, tomato gray mold and pepper anthracnose and has small using amount, low using cost for peasants, wide usable range, and the like compared with the traditional medicament.