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The inhibitory effect of barley straw (Hordeum vulgare) on cyanobacteria has been observed in many field and laboratory studies for over 30 years, although the compounds responsible for this anti‐cyanobacterial effect have remained unknown. In this study, a pair of chiral flavonolignans were isolated from barley straw extract using a bioassay‐guided isolation procedure against Microcystis sp. The structures of the allelopathic compounds were elucidated by NMR (nuclear magnetic resonance) and HPLC‐MS (high performance liquid chromatography‐mass spectrometry), and turned out to be salcolin A and B. The enantiomers differ in their anti‐cyanobacterial abilities. Both enantiomers exhibited inhibitory effects on Microcystis sp., and the EC₅₀ (concentration for 50% of maximal effect) of salcolin A and B were 6.02 × 10⁻⁵ and 9.60 × 10⁻⁵mol l⁻¹, respectively. Furthermore, the modes of actions of the …