The chemical composition of extractions of different soil layers from a Rhododendron irroratum forest was analyzed to provide basic data on the relationships between the community's natural regeneration barrier and allelopathy and to explain the natural regeneration barrier factors from the perspective of chemical ecology. The allelopathic effects of litter layer (L layer), humus layer (H layer), and soil layer (S layer) extractions were compared using seed germination methods. The organic compounds in the three extractions were assessed using the internal standard method with gas chromatography-mass spectrometry (GC/MS). (1) The seed germination test showed that of the three layers, L layer had the strongest inhibitory effect and its extraction inhibits seed germination, while H layer and S layer had some inhibitory effect that was not significant compared with the control. (2) Thirty-one components were identified from the organic compounds of L layer, H layer, and S layer, in which there were 6, 8, and 8 kinds, respectively, with relative contents of more than 5%. The main allelochemical components of L layer are glycerol and palmitic acid, with relative contents as high as 19.56% and 19.17%; the main allelochemical components of H layer are 2-hydroxypropanoic acid and palmitic acid, with relative contents up to 14.05% and 12.48%; and the main allelochemical components of L layer are palmitic acid and 2-hydroxyacetic acid, with relative contents as high as 14.91% and 12.79%. L layer had the highest contents of allelochemical compounds, and L layer was the main source of soil allelochemicals. The main allelochemicals in H layer and S layer were long-chain fatty acids and organic acids, and the major allelochemicals in L layer were long-chain fatty acids and alcohols. The allelochemicals of Rhododendron community soils might be one of the main reasons for the natural regeneration barrier in this community.