Allelopathy is a biological process in which one organism releases biochemicals that affect the growth and development of other organisms. The current investigation sought to determine the allelopathic effect of Rumex acetosella on white clover (Trifolium repens) growth and development by using its shoot extract (lower IC50 value) as a foliar treatment. Here, different concentrations (25, 50, 100, and 200 g/L) of shoot extract from Rumex acetosella were used as treatments. With increasing concentrations of shoot extract, the plant growth parameters, chlorophyll and total protein content of Trifolium repens decreased. On the other hand, ROS, such as O2.− and H2O2, and antioxidant enzymes, including SOD, CAT, and POD, increased with increasing shoot extract concentration. A phytohormonal study indicated that increased treatment concentrations increased ABA and SA levels while JA levels were reduced. For the identification of allelochemicals, liquid‒liquid extraction, thin-layer chromatography, and open-column chromatography were conducted using R. acetosella shoot extracts, followed by a seed bioassay on the separated layer. A lower IC50 value was obtained through GC/MS analysis. gammaSitosterol was identified as the most abundant component. The shoot extract of Rumex acetosella has strong allelochemical properties that may significantly impede the growth and development of Trifolium repens. This approach could help to understand the competitive abilities of this weed species and in further research provide an alternate weed management strategy.