Crops with weed suppressive root exudates or the direct use of bioherbicidal allelochemicals is a new approach in integrated weed management systems. In this context, the allelopathic activity and chemical composition of root exudates from six genotypes (modern varieties and landraces) of barley were characterized. The phenolic acids appeared to be particularly implicated in the inhibitory action of barley root exudates against Bromus diandrus. The amount of these compounds was higher in sandy substrate than in sandy-clay-loam substrate. Ten phenolic acids and one phenylpropanoid derivative were present, in addition to saponarin, a newly identified flavonoid in barley root exudates. Seven compounds explaining variability in the inhibitory activity of barley roots (stepwise analysis) and one compound detected only in highly allelopathic genotypes were toxic against receiver plants. Most compounds had a greater inhibitory effect on the growth of great brome than the barley genotypes. The synergistic and/or additive effect of the eight compounds appeared to be the source of the toxicity. Benzoic acid, the mixture of compounds, saponarin and salicylic acid were the most efficient compounds against the great brome and the less aggressive against barley. Overall, the results revealed the allelopathic potential of the water-soluble compounds exuded by the roots of living barley plants. These compounds included saponarin, a flavonoid not yet recognized as a barley root allelochemical.