In a previous study, lavender (Lavandula spp.) was found to be highly phytotoxic towards annual ryegrass (Lolium rigidum, ARG), a major weed of winter wheat crops in Australia. This research aimed to further explore this relationship and determine the chemical(s) responsible for the observed effect. In bioassay, it was determined that the stem and leaf extract of L. x intermedia cv. Grosso ranked highest and had the potential to reduce significantly the root growth of several plant species. An extract concentration of 10% almost completely inhibited ARG root growth. When the extract was tested for stability, there was no loss in phytotoxicity after the 256 day trial. Via bioassay-guided fractionation and chromatographic techniques, it was determined that the sub-fraction consisting of coumarin and 7-methoxycoumarin was most phytotoxic towards ARG. Chemoassays of 18 structural analogues of coumarin showed that coumarin itself was the most phytotoxic and largely responsible for the observed phytotoxicity of the extract. Soil trials were conducted using pure coumarin and the lavender extract, and in both instances, shoot length and weight were significantly reduced by post-emergence application at all concentrations evaluated.