The prevailing study is an attempt to utilize Methylisothiazolinone (MIT) which was applied efficiently in other applications away from fabric finishing fields such as preservation and health care. A certain concentration of the substance were applied to the scoured woven cotton fabric , the use of distinct methods of treatment which includes Exhaustion and pad-dry-Thermo-fixation. The results obtained showed that using pad-dry-Thermo-fixation gave better nitrogen content more than exhaustion method, subsequently we continued the work using padding method which gave more anti-bacterial conduct . Furthermore, by varying the different factors influencing the reaction such as MIT's concentration (10-50 mg/l or ppm), curing time (1-10 min.) and curing temperature (120-170°C) the study reached the optimum conditions to get the best anti-bacterial treatment for cotton fabrics against gram positive bacteria(Staphylococcus Auras) and gram negative bacteria ( Escherichia Coli) . FT-IR spectroscopy, Antibacterial test according to (AATCC) and SEM were used to assure the obtained results.