The role of different growth media and chemical enhancer on synthesis of secondary metabolites Cladosporium resinae (NRL-6437) was investigated for their in vitro biological activities. Cladosporium resinae (NRL-6437) were grown in various nutrient media (Czapeak-dox Broth (CB), Czapeak Yeast-extract Broth (CYB), Yeast Extract Sucrose (YES), Potato Dextrose Broth (PDB) and Czapeak-dox (supplemented with glucose and starch) Broth (CGSB) for the production of metabolites. Two chemical epigenetic modifiers (suberoyl-anilide hydroxamic acid (SAHA) and 5-azacytidine (5-AZA) were also used for the expression of silent genes for secondary metabolite production. Our results indicated that among different media, Czapeak yeast extract broth produced more secondary metabolites. Application of 15mM of both modifiers was effective for the expressions of silent genes resulting in an increased metabolites production. Secondary metabolites extracted in ethyl acetate and fractionized in n-Hexane were also tested for their biological activity. The secondary metabolites revealed varying degrees of growth inhibitions of the tested organisms. Similarly, these metabolites were also active against brine shrimps and Lemna.