Abstract : The high level of resistance to pathogenic bacteria causes the number of antibiotics to be limited. Therefore, searching for new antibiotics that can kill these resistant bacteria is necessary. A new approach to obtaining new metabolic products with various structures is "One Strain Many Compounds" (OSMAC). This strategy can offer a quick, easy, and efficient way to increase the diversity of secondary metabolites by enhancing and activating "silent biogenetic gene clusters." Through this research, the secondary metabolites of Penicillium simplicissimum Ch06 in a solid rice medium were compared to the same medium with the addition of 3.5% MSG and 3.5% NaNO3. The content of secondary metabolites was analyzed using LC-MS/MS method. The antibacterial activity of the fungus extracts was tested using the agar diffusion method. Adding MSG and NaNO3 to rice media affected the formation of secondary metabolites from the fungus. The antibacterial activity of fungal extracts induced by adding MSG and NaNO3 was greater than that of the control medium. The fungal metabolite profiles analyzed by liquid chromatography(LC)-mass spectrometry (MS) showed a clear difference of several peaks in the LC profile following the same trend with the variation of the antibacterial activity of the fungal extracts. Our results showed a clear difference in cultivation media with the addition of MSG / NaNO3 and fungal bioactivity.