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Fistula in Ano is a common anorectal condition in populations and its prevalence is second highest after (hemorrhoids). Ksharasutra is one of the chief treatment modality in the management of fistula in ano. Main objectives of this study; clinical efficacy and safety of the Ksharasutra treatment in the management of fistula in Ano, identification of antimicrobial activity and study the chemical composition of Ksharasutra. Safety of the Ksharasutra treatment evaluated by using anal manometer. Anal incontinence was assessed by using incontinence questionnaire. Quality of Life evaluated by using Gastrointestinal Quality Life index (GIQLI). Histopathology study, monitoring the Symptom score and Unit Cutting Time was used to evaluate the clinical efficacy of Ksharasutra treatment. The biochemical tests were carried out to identify the microorganism in chronic fistula. To determine the antibacterial activity of Kshara, Ksharasutra, Curcuma longa, Euphorbia antiquarum latex and Comphiphoramukul were carried out by using Kirby baurer Disc diffusion and Well diffusion methods.