Collagenase was found to be the most important enzyme, produced by mycotic keratitis fungi. Therefore, Aspergillus flavus collagenase enzyme has been purified by ammonium sulphate precipitation, Sephadex G25 and DEAE-cellulose chromatography. Electrophoretic analysis for the purified enzyme indicated one subunit of molecular weight of 70-90 KDa when examining on SDS-PAGE. Cetrimide (cetyl trimethyl ammonium bromide) has been tested against the purified collagenase enzyme and indicated reversible competitive inhibitor (kis = 0.15 mg/ml) with high promising activity. Cetrimide might be used to inhibit mycotic keratitis fungi.