The search for antifouling compounds led us to investigate the production of the secondary metabolite meleagrin by fermentation of Penicillium rubens Biourge. Since initial growth attempts yielded none of the desired product, subsequent growth condition studies sought to enhance meleagrin production. The aim of this study was to identify conditions that selectively enhanced the production of meleagrin and other desirable secondary metabolites. This goal was accomplished by detecting, isolating and characterizing the secondary metabolites resulting from alterations to the growth media’s composition as well as the addition of a biosynthetic precursor, roquefortine C. In addition to identifying growth conditions that produce meleagrin, the present study optimized the production increase production of meleagrin by 446%, viridicatin by 237%, penitrem E by 344%, and penitrem A by 481%. This research represents the first systematic growth enhancement study for P. rubens Biourge and illustrates that slight modifications to growth conditions can have a significant impact on the production of secondary metabolite distribution and abundance.