Many marine natural products hold great potential for the development of new and much needed drugs. However, the production of active metabolites by marine-derived microorganisms is usually very low, and large-scale culture has to be involved to meet the need of chemical structural modification and deep pharmacy study. In order to enhance the production of a novel cytotoxic sulfur-containing chromone oxalicumone A (OA), germinating spores of a marine-derived wild strain Penicillium oxalicum SCSGAF 0023 were mutated by microwave and ultraviolet light irradiation, which led to the obtainment of a mutant P. oxalicum SCSIO 24-2 that could produce fivefold increase in OA production (3.42 ± 0.21 mg/l) as compared to the wild strain. This is the first report that germinating spores are applied in marine-derived Penicillium sp. mutating to enhance the production of OA. Further, Plackett-Burman design and central composite design were adopted to optimize the basic medium components for increasing OA production by the mutant SCSIO 24-2 in shake flasks. The results indicated that three medium components including mannitol, maltose, and L-cysteine had significant effects on OA production, and their concentrations were optimized as 36, 27.9, and 0.99 g/l, respectively. In the optimized medium, the OA production (18.31 ± 0.27 mg/l) by mutant SCSIO 24-2 was 4.4-fold higher than that in the basic medium. These results of this work promise to improve the present production of OA and may be adopted to enhance other objective products' production by marine-derived fungi.