This study aimed to characterization and evaluates the effectiveness of red dragon fruit (Hylocereus polyrhizus) peel extracts as an antimicrobial agent. Red dragon fruit peel extracts were obtained by maceration using solvent at pH 5. Phytochemical characteristics, total phenols, antioxidant, and antimicrobial activity of the peel extracts were observed. Antioxidant and antimicrobial activities of the extracts were associated with high phytochemical compounds and total phenols contained in the extracts. Red dragon fruit peel extracts with various percentages prepared and their physicochemical characteristics, nutrients, antioxidant activity, and microbiological profile were analyzed. The current study is to evaluate the susceptible antimicrobial activity of crude 50% hydro alcoholic Peel Extracts of Red Dragon Fruit (Hylocereus polyrhizus) against different clinical isolates (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, A. niger and Candida albican) of microbial disease so as to trip up on the other alternatives and overcome the upcoming era of increasing microbial resistance. Furthermore, the results of the In vivo assay on thirty mice model showed that the treatment of mice with the 100mg/ml of aqueous extract and Chloramphenicol have more effectiveness than other concentrations and the number of bacteria P. aeruginosa was reduced significantly (P ≤ 0.001) during the treatment days. Results showed that the red dragon fruit peel extracts have an Antimicrobial Activity.