This study presents the degradation of phenanthrene by Mycoplana sp. MVMB2 isolated from petroleum contaminated soil and the media optimization by factorial design experiments. The Plackett–Burman design was used to evaluate the effects of eight variables (potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, glucose, inoculum concentration, and phenanthrene concentration) on phenanthrene degradation. Based on the results, the critical medium components having significant influence on the degradation were found to be disodium hydrogen phosphate, magnesium sulfate, ferrous sulfate, and phenanthrene. Furthermore, these four variables were used as central composite design parameters. The optimum minimal salt medium composition obtained by conventional and factorial design experiments for the degradation of phenanthrene by Mycoplana sp. MVMB2 at pH 6.5 and 30°C were found to be, potassium 2.5 g/L dihydrogen phosphate, 0.3505 g/L disodium hydrogen phosphate, 0.5501 g/L magnesium sulfate, 0.02 g/L calcium chloride, 0.0261 g/L ferrous sulfate, 0.6756 g/L phenanthrene, 0.5 g/L glucose, 0.5 g/L ammonium sulfate, and inoculum 5% v/v. The phenanthrene degradation was confirmed by analyzing the metabolites formed.