Mespilus germanica as a valuable medicinal plants used in traditional medicine. Aim of this study, investigated effect of combination various ratio of solvents (Glycerin, Ethanol, Methanol and Water) on the efficiency of Mespilus extract by mixture optimal design. Numerical optimization was used to obtain the optimal formulation of solvent. At the end of the day, the antimicrobial effect of Mespilus extracts based on three methods (agar diffusion Method, Minimum Inhibitory Concentration and Minimum Bactericidal Concentration) on the three microorganisms managing infectious diseases was investigated in vitro. In this study, investigated effect water, ethanol, methanol and glycerin on the five levels (0, 31.25, 83.33, 125,250 mm) on efficiency of Mespilusextracts by mixture optimal design. Diffusion agar test, Minimum Inhibitory Concentration, Minimum Bactericidal Concentration by microbroth dilution method was used to determination Susceptibility of bacterial isolate. The Result indicated that Scheffe polynomial model was highly significant for efficiency of Mespilusextracts. Minimum Inhibitory Concentration of Staphylococcusaureus, Escherichia coli, Pseudomonas aeruginosa were 1.25, 2.5 and 5 mg/ml, respectively. The optimum condition has been found as following: glycerin (0 ml), water (23.7 ml), methanol (100.2ml) and ethanol (126.1 ml) respectively. It’s worth to mention that there was no significant difference between experimental and predicted value in optimum condition. Mespilus extract was highly significant for reduce of Infectious bacteria. Mixture methodology based on the D-optimal design was able to statistical assessment extraction process with the minimum experiment.