<p>A new natural mycotoxin was isolated from the fermentation broth of <i>Trichoderma</i> sp. Jing-8 and the structure was determined as alternariol 1′-hydroxy-9-methyl ether (<b>1</b>), together with twelve known compounds. The structures were elucidated on the basis of their 1D, 2D NMR spectra and mass spectrometric data. Compounds <b>1</b>, <b>8</b> and <b>9</b> indicated inhibitions against germination of the seeds of cabbage with MICs < 3 μg/mL. The compound <b>1</b> showed the antibacterial activity against <i>Bacillus subtilis</i> and <i>Staphylococcus aureus</i> with MICs at 64 μg/mL. Compound <b>1</b> and <b>3</b> showed significant DPPH radical-scavenging activities with IC<sub>50</sub> at 12 μg/mL, respectively. The OH at C-1′ in compound <b>1</b> decreased the cytotoxicity of these mycotoxins. A primary structure–activity relationship about the alternariol derivatives was discussed. Compounds <b>2</b>–<b>7</b> and <b>8</b> were the first time to be isolated from the <i>Trichoderma</i>.</p