The search for bioactive substances has propelled studies in underexplored new natural sources, such as marine organisms and their endophytic fungi. Microorganisms, especially those obtained from marine sources, are important for the production of metabolites with high structural diversity and therapeutic potential. In this context, this study aims to establish cultivation conditions for optimal extracts yields of one endophytic fungus strain through chemometric tools (Factorial Planning). Annulohypoxylon stygium was isolated from the red algae Acanthofera spicifera and investigated for the effects of such parameters on the production of secondary metabolites and samples bioactivity. Such approach is aimed to optimising subsequent experimental steps as isolation of new bioactive compounds, in addition to contributing to a deeper understanding on the chemistry of marine species associated microorganisms .