The inhibitory effects and mechanisms of eugenol were studied in pathogenic fungi that were isolated from passion fruit affected with naturally occurring fruit rot; additionally, the effect of eugenol treatment on fruit rot resistance in passion fruit was investigated. The corresponding results demonstrated that the pathogenic fungus causing passion fruit rot was Lasiodiplodia theobromae and that the minimal inhibitory concentration of eugenol against L. theobromae was 0.30 mg/mL. Eugenol significantly inhibited mycelial growth and spore germination of this fruit rot fungus. Further, nucleic acid release, electrical conductivity, and protein and soluble sugar content of the fruit rot fungus gradually increased with increasing eugenol concentrations. Propidium iodide staining revealed that the cell membrane integrity of L. theobromae hyphae was disrupted when treated with eugenol. In addition, eugenol treatment inhibited the spread of disease spot diameter after inoculation with L. theobromae, effectively increased APX, SOD, CAT, POD, 4CL, C4H, and PAL activities, promoted the accumulation of disease-stage-related proteins CHI and GLU, and increased the total phenol and flavonoid content during storage in passion fruit. Overall, these results suggest that eugenol has good application prospects for the effective control of fruit rot in passion fruit.