The aim of this study was to separate or purify some bioactive compounds from flowers of alfalfa(Medicago sativa L.) and to test of the isolated compounds on alfalfa for their autotoxicity and on Echinochloa crus-galli for their allelopathy for seed germination and seedling weight. Using thin layer chromatography(TLC) of CHCl₃ extracts, the most inhibitory band to alfalfa seed germination was determined. Germination inhibition of this extract suggested a complex chemical interaction. Separation and purification of compounds with CHCl₃ extract of fresh alfalfa flowers were conducted by a silica gel TLC, and microcrystalline cellulose TLC (MCTLC), followed by droplet countercurrent chromatography(DCCC) bioassay. Preliminary identification was done lay high performance liquid chromatography(HPLC) on the most inhibitory fractions in DCGC. Ferulic acid, caffeic acid, vanillic acid, rutin, narringin were identified in fraction 5 and ferulic acid, caffeic acid, vanillic acid, rutin, coumarin in fraction 6. The phytotoxicity of their individual compound was tested on alfalfa and Echinochloa crus-galli seed germination and seedling weight. Coumarin and ferulic acid showed the most inhibitory effect on alfalfa seed germination and Echinochloa crus-galli seedling fresh and dry weight. These compounds may be, at least in part, involved in autotoxicity and allelopathy.