This research aimed to assess the biodegradation potential of phenanthrene in a simulated aqueous solution using an isolated bacterial strain, Rhodococcus sp., with the assistance of a Box-Behnken design matrix. To gauge the impact of key operational factors, pH (6.0 to 9.0), temperature (20 to 40°C), initial phenanthrene concentration (50 to 100 ppm) and incubation time (7 to 21 days), the Response Surface Methodology (RSM) was employed to design the biodegradation experiment. The experimental findings highlighted incubation time as the most influential variable followed by initial phenanthrene concentration, pH and temperature via Box-Behnken Design (BBD) matrix. To determine the maximum phenanthrene biodegradation, the Desirability Function Methodology (DFM) was applied. Within the designated parameter ranges, the highest phenanthrene biodegradation (70.0%) was observed at pH 7.3, temperature of 30°C and an initial phenanthrene concentration of 70 ppm on the 19th day of incubation. The model's fitness was thoroughly validated, with a correlation coefficient (R²) of 0.9899 and a "Prob>F" value below 0.0500, affirming its suitability for optimization purposes. This study underscores the utility of the RSM in optimizing operational variables, ultimately resulting in significant time and cost savings for experimentation.