In greenhouse experiment, the allelopathic effects of 6-barley genotypes were assessed on the morphological features of weeds, Bromus diandrus Roth. and Stelleria media L. The inhibitory effects of root exudates depended on the barley genotype and the physico-chemical properties of the soil, and their interactions. The sandy soils with low organic matter and nutrients content showed more the allelopathic potential. A predictive model of the allelopathic activity of barley was proposed based on soil properties and tested weeds. Overall, the stepwise model showed that the content of phenolic acids was the major determinant of allelopathic activity, besides the soil chemical characteristics (electrical conductivity and carbon and sodium content). Soil microbial communities decreased the allelopathic activity of barley. Drainage and aeration might explain the slightly higher inhibitory activity in a non-autoclaved sandy substrate than a clay-loam substrate. When recommending allelopathic barley genotypes for cultivation, the environmental factors, physico-chemical properties of soil and rhizosphere microbiome might reduce or enhance their allelopathic potential.