Antimicrobial activity of Morinda citrifolia L., extract on typical pathogenic bacteria and its application in Pangasius hypophthalmus fillet were carried out. A broth dilution method was employed to determine the minimum inhibition concentration (MIC) of the extract on Staphylococcus aureus and Escherichia coli O157:H7. The MIC was found to be 200 mg and 400 extract powder/mL for E.coli O157:H7 and S.aureus, respectively. Application of the extract at MIC points into the fish fillet at the cleaning stage with various soaking time (0, 5, 10, 20 and 30 min), E. coli O157:H7 manifested more sensitive to the extract than S. aureus by the decrease of the numeral population, approximately 2.38 log cfu/g and 2.18 log cfu/g respectively after soaking within 5 min. The presence of essential antimicrobial compounds, containing in Morinda citrifolia L., extract in powder form such as Anthraquinone, Coumarine and Iridoid, resulted in antimicrobial efficacy of Morinda citrifolia L., extracts and also contributed to the potential usage as a natural preservatives in food industry, particularly fish fillet product, and food safety aspects.