Optimization based on appropriate parameters can be applied to improve a process. Mazut degradation as a critical issue in environment requires optimization to be efficiently done. To provide biodegradation conditions, experiments were designed on the least interactions among levels of parameters consisting of pH, Tween 80, glucose, phosphorous source, nitrogen source, and time. Kinetic constants and biomass were calculated based on 16 assays, designed using Taguchi method, which constructed various mazut biodegradation conditions. Kinetics of mazut degradation by newly isolated bacteria Enterobacter cloacae closely followed second order kinetic model. Results of the 16 experiments showed that biomass was in the range of 0.019 OD₆₀₀ to 2.75 OD₆₀₀, and biokinetic constant was in the range of 0.2 × 10^-5 L/ (mg day) to 10^-4 L/ (mg day). Optimal level for each parameter was obtained through data analysis. For optimal biomass equal to 2.75 OD₆₀₀, optimal pH, Tween80, glucose, phosphorous source, and time were 8.3, 4 g/L, 4 g/L, 9 g/L, and 10 days, respectively. For biokinetic constant equal to 1.2 × 10^-4 L/ (mg day), optimal pH, Tween80, glucose, phosphorous source, and nitrogen source were 8.3, 1 g/L, 4 g/L, 1 g/L, and 9 g/L, respectively. The optimum levels for biomass and biokinetic constant were the same except the levels of the Tween 80, and phosphorous source. Consequently, mazut may be more degraded with adjusting the conditions on the optimum condition.