Abstract: This study investigated the potential application of sucrose esters (SEs) derived from Nicotiana glutinosa for biopesticide development. SEs were extracted using solvent extraction, purified by normal-phase silica gel column chromatography, followed by identification and quantification using GC/MS analysis. The antifungal properties against six phytopathogenic fungi was evaluated through the mycelial growth rate method, and the anti-tobacco mosaic virus (TMV) activity using the half-leaf lesion assay. Two distinct SE configurations were isolated and identified from N. glutinosa: SE-II (acetylated at fructose C-3′) and SE-III (non-acetylated at fructose C-3′). SEs contents varied significantly among tissues, with the highest levels observed in upper leaves (3.56 mg/g fresh weight) and flowers (3.21 mg/g), substantially exceeding those in middle leaves, lower leaves, and stems. A progressive decrease in the SE-II/SE-III ratio was noted from lower to upper plant positions. The crude SE extract, SE-II, and SE-III exhibited broad-spectrum antifungal activity, with notably strong inhibition observed against Botrytis cinerea. At a concentration of 20 μg/mL, three SE forms achieved inhibition rates of 57.75%, 65.58%, and 84.73%, respectively. The antifungal activity of the crude SE extract against B. cinerea was superior to the botanical fungicides eugenol and osthole. SE-II showed comparable activity to the synthetic fungicide pyrimethanil, whereas SE-III exhibited significantly stronger inhibitory effects than pyrimethanil. In anti-TMV assays at 200 μg/mL, inhibition rates were 36.33% (crude extract), 69.09% (SE-II), and 64.41% (SE-III). The antiviral activity of the crude SE extract matched that of oligosaccharins, while SE-II and SE-III showed efficacy comparable to that of ningnanmycin. These findings identify N. glutinosa represents a valuable source of diverse and bioactive SEs showing promising potential for the management of biofungicides against gray mold and antiviral agents for crop protection.