We studied the polyphenol (phenolic compounds and flavonoids) composition and allelopathic effects of Acacia melanoxylon R. Br. aerial foliage aqueous extract (0%, 25%, 50%, 75% and 100%) on the seedling growth and plant biomass of the general biotest species, lettuce (Lactuca sativa). Mean leaf fresh weight, leaf dry weight, root fresh weight and root dry weight were decreased following exposure to Acacia aerial foliage, flowers aqueous extract (AFE) and phyllodes aqueous extract (APE) after 6 days. The reduction in plant dry biomass was more than 50% following treatment with AFE. The decrease in mean root length was approximately 37.7% and 29.20% following treatment with Acacia flowers extract (AFE) at 75% and 100% concentration, respectively. Root dry weight of L. sativa was reduced by both flowers and phyllodes extract. The reduction of protein contents in lettuce leaves following Acacia foliage extract proved that both AFE and APE exhibit polyphenols that causes the toxicity which led to decrease in leaf protein contents. High-Performance Liquid Chromatography (HPLC) was employed to analyze the A. melanoxylon flowers and phyllodes. A total of 13 compounds (accounting for most abundant compounds in flowers and phyllodes) include different flavonoids and phenolic compounds. The phytochemical compounds detected were: Gallic acid, protocatechuic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillic acid, syringic acid, p-coumaric acid, and ferulic acid. The major flavonoid compounds identified include rutin, luteolin, apigenin, and catechin. Allelopathic effects of flower and phyllodes extracts from A. melanoxylon may be due to the presence of above compounds identified by HPLC analysis.