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Background To enhance discovering efficiency of new herbicidal compounds, a strategy for rapidly screening out strongly herbicidal chemical components from natural resources is necessary. Results Seventeen essential oils selected from 14 Eucalyptus species and hybrids were evaluated for their herbicidal activities on annual ryegrass. A feasible strategy was established for rapidly pinpointing the chemicals contributing to strong herbicidal activities without the process of isolation and purification of individual compounds. This strategy was based on gas chromatography-mass spectrometry (GC-MS) coupled with principal component analysis (PCA) and verified by the bioassay results using several individual compounds. Two strong herbicidal compounds trans-pinocarveol and α-terpineol were screened out using the strategy. The strong herbicidal activity of trans-pinocarveol was discovered for the first time. Conclusion The established strategy of screening herbicidal compounds from natural resources is simple, feasible and reliable. © 2019 Society of Chemical Industry.