The invention discloses a preparation method of a water-resistant antimicrobial edible starch film. The preparation method comprises accurately weighing an appropriate amount of a mixture of starch and octenyl succinic starch ester, putting the mixture into deionized water, stirring the mixed solution to obtain a suspension solution, heating the starch suspension solution in a boiling water bath for 15min, continuously stirring the mixed solution so that the mixed solution is fully gelatinized, adding a certain amount of glycerol into the paste, carrying out heating stirring for 10min, cooling the starch paste to 45 DEG C, adding a certain amount of emulsified oregano essential oil into the paste, carrying out uniform mixing under high rate stirring, carrying out degassing in vacuum for 30min, pouring the starch paste to a flat plate, paving the starch paste, drying the starch paste at a certain temperature and stripping off the dried paste to obtain the water-resistant antimicrobial edible starch film. The preparation method utilizes combination of potato starch having excellent film formation performances and octenyl succinic starch ester, utilizes glycerin as a plasticizer and utilizes oregano essential oil so that antibacterial properties of the edible film are greatly improved. The preparation method is safe and environmentally friendly and has a good market potential.