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There is currently much interest in finding new phytochemicals among plants and fungi as nature-based alternatives to replace problematic herbicides such as glyphosate, which are preferentially used in agricultural production. We discovered striking herbicidal potency in <i>Cordyceps militaris</i> (L.) and identified cordycepin as its principal plant growth inhibitor. Cordycepin obtained as an ethyl acetate extract was subjected to column chromatography and evaluated for its bioassay-guided phytotoxic capacity against <i>Raphanus sativus</i> (radish), showing a maximum inhibition on germination and growth of radish (IC₅₀ = 0.052&#8722;0.078 mg/mL). Gas chromatography-mass spectrometry (GC-MS) (<i>m</i>/<i>z</i>: 251.2) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) ([M + Na]⁺ <i>m</i>/<i>z</i>: 274.1; [M + H]⁺ <i>m</i>/<i>z</i>: 252.1) analyses confirmed cordycepin as the major component of the tested column fraction (55.38%). At 0.04 mg/mL, cordycepin showed 3.8&#8722;5.9- and 3.3&#8722;3.7-fold greater inhibition of the germination and growth of radish than benzoic acid (BA) and glyphosate, respectively. Compared with BA, isolated cordycepin reduced plant chlorophyll and carotenoid contents (2.0&#8722;9.5 -fold), while proline, total phenolic and total flavonoid contents were increased 1.2&#8722;1.8-fold. Finally, cordycepin promoted electrolyte leakage and malondialdehyde accumulation in radish aerial parts. Thus, cordycepin successfully isolated from <i>Cordyceps militaris</i> is a highly potent plant growth inhibitor with pending worldwide patent and may become a potential plant-based novel alternative to the disputed glyphosate.