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Equisetin was isolated from endophytic fungus <i>Fusarium</i> sp. JDJR1 via bioassay-guided isolation, and it was evaluated as an anti-microbial and herbicidal agent. Bioactivity assessments revealed that equisetin exhibited a broad spectrum of fungicidal and anti-bacterial activity against various plant pathogens. The EC₅₀ values against <i>Botrytis cinerea</i>, <i>Fusarium graminearum</i>, <i>Sclerotinia sclerotiorum</i>, and <i>Rhizoctonia solani</i> ranged from 10.7 to 21.0 μg/mL. Notably, equisetin demonstrated activity against <i>Xanthomonas oryzae</i> pv. <i>oryzicola</i>, <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> and <i>Pseudomonas solanacearum</i> with an MIC range of 4–16 μg/mL, surpassing the efficacy of the positive control: streptomycin sulfate. Equisetin, at a concentration of 100 μg/mL, could completely inhibit the spore germination of <i>F. graminearum</i>. In in vivo protection experiments, the protective efficacy of equisetin against <i>B. cinerea</i> on tomato fruits at 200 μg/mL reached 72.9%. Furthermore, in herbicidal activity tests that use the Petri dish bioassay method, equisetin had a good inhibitory effect on the root growth of weeds. At a concentration of 100 μg/mL, the inhibition rates for <i>Echinochloa crusgalli</i> and <i>Eclipta prostrata</i> root were 98.8 and 94.4%, respectively.