IN THIS STUDY, we established of an application protocol for the micropropagation of Dillenia indica with an estimate of betulinic acid from callus, leaf, and regeneration plant. Moreover, the antimicrobial and cytotoxic efficacy of the crude extract and pure compound was compared. The highest number of shoots (3.667 shoots), leaf number per jar (27 leaves) and shoot length (3.667cm) were recorded from the MS medium containing 2.0mg L-1 of benzyl amino purine (BAP). Moreover, different physical elicitors (ultraviolet irradiation, microwave radiation, and light quality) were used on the production and increased the concentration of betulinic acid in callus of D. indica. The data revealed that the highest betulinic acid content (341.775mg/ 100g fresh weight) was recorded in callus microwaved with 200 watts for 10s. Which was three times more than the betulinic acid content recorded in the mother plant. In addition, different crude extracts of D. indica were used to determine their biological activity against six microorganisms. The data showed that the highest diameter of inhibition zones (17.0 mm) was recorded in callus culture extractions incubated under blue light with Candida albicans. The cytotoxic efficacy of betulinic acid and extract of callus, mother plant leaf or in vitro regeneration plants of D. indica were investigated against the Vero cell line by MTT. The results revealed that The IC50 values were 115.7 ± 9.17, 114.98 ± 5.2, 59.45 ± 3.06, and 72.76 ± 6.93μgmL-1 of betulinic acid, callus extract, leaf, and regeneration plant, respectively.