Lipase-mediated polymer degradation is a robust alternative approach to conventional methods due to biocompatibility and mild conditions. In the present study, response surface methodology was applied to improve the production of lipase from Penicillium fellutanum by optimization of various process parameters. Under the optimized bioprocess conditions of pH 5.0, incubation time 24 h, temperature 35 °C, and lactose as an additional carbon source in 40 experimental runs, the maximum lipase titer of 1038.86 U/gds was achieved, 2.05-fold higher than the lipase yield in basal medium. All the linear and interactive coefficients (except linear carbon source) were found significant by analysis of variance. The as-synthesized cell-free lipase extract was partially purified by ammonium sulfate fractionation and dialysis (2.06-folds, 272.37 U/mg proteins) and applied to the degradation of polyester vylon 200. The biocatalytic action of the enzyme results in an 81% weight loss of PV-200 after 7 days of incubation at pH 7.5 and 35 °C. Different characterization techniques, i.e., SEM, FT-IR, and DSC corroborated the lipase-catalyzed degradation of PV-200. The untreated polyester film had a smooth surface, while after enzymatic treatment, deformities and various micron-sized holes and cracks appeared on the film surface. In conclusion, the outcomes of study display a high potential of lipase as green and ecofriendly biocatalyst for efficient degradation and depolymerization of polyester for environmental safety. Graphic Abstract