Botanical fungicides comprise attractive alternatives to chemical fungicides because of their environmental compatibility. Flavonoids extracted from Tagetes erecta L. have an inhibitory effect on fusarium wilt in watermelons caused by Fusarium oxysporum f. sp. niveum (FON). In this study, we synthesized one of these flavonoids, 2,5-dicyclopentylidene cyclopentanone (Tagetes erecta L. fungicide (TEF)) and assessed its activity against FON. In vitro, TEF inhibited FON growth and killed FON cells directly. TEF also affected FON cell physiology and mycelial structure. In watermelon plants with fusarium wilt, TEF protected the leaf cell structure and improved the germination rate of infected seeds while increasing overall plant resistance. A TEF-resistant mutant (FONM) was created by chemical mutagenesis. FON and FONM were analysed using iTRAQ and RNA-Seq, which identified 422 differentially expressed proteins and 7817 differentially expressed mRNAs in the proteome and transcriptome, respectively. The FONM mutations caused changes in the cell membrane and cell wall, which may constitute the site of action of TEF. Together, these results demonstrate that TEF could effectively control the watermelon fusarium wilt caused by FON, possibly through the inhibition of sterol biosynthesis. The data presented here suggest that TEF represents a new potential botanical anti-fungal drug.