Background: Prodigiosin is a bioactive bacterial pigment produced by Serratia marcescens and possesses anticancer, antibiotic, antifungal, antibacterial, and immunosuppressive activities without any toxic effects on human cells. Objectives: The study was planned to optimize the effect of ultravoilet (UV) light and acridine orange (AO), as mutagens and incubation time on prodigiosin production by S. marcescens in nutrient agar broth and peptone glycerol broth. Materials and Methods: The optimization was done by constructuing a quadratic polynomial response surface model using a bifactorial central composite design. The S. marcescens starian was treated with UV light (0, 30, 60, 90, and 120 s) and various concentrations of AO (0, 50, 100, 150, and 200 μg/mL) and incubated in the selected growth media for different times (0, 15, 30, 45, and 60 h). The prodigiosin production was measured in terms of change in pH and optical density (OD) of the ethanolic extracts of culture media. Results: The prodigiosin production was found to be a significant linear and quadratic function of incubation time in both of the culture media. However, a statistically non-significant increased prodigiosin production, in terms of pH and OD of the extracts, was observed under the influence of both mutagens. The optimum values of incubation time to achieve optimal production of prodigiosin from UV light-exposed and AO-treated cultures were found to be in the range of 37–60 h, respectively. Conclusion: The pigment production was increased significantly with an increase in the incubation time. The study provides the optimum conditions for the production of prodigiosin in research and commercial field.