Metabolomics has become an important tool in the search for bioactive compounds from natural sources, with the recent inclusion of marine organisms. Of the several steps performed in metabolomics studies, the extraction process is a crucial step—one which has been overlooked for a long time. In the presented study, a pressurized liquid extraction system was used to investigate the effect of extraction parameters such as pressure, temperature, number of cycles, and solvent polarity on the chemical diversity of the extract obtained from the marine sponge, Xestospongia. For this, a full factorial design (24) was performed using a chemical diversity index, which was found to be a suitable tool to determine the efficiency of the extraction process, as the response variable. This index was calculated using a logarithmic transformation of 1H NMR signals. Three factors (number of cycles, temperature, and solvent polarity) and two interactions were found to affect the chemical diversity of the obtained extracts significantly. Two individual factors (temperature and solvent polarity) were selected for further study on their influence on sponge metabolites using orthogonal partial least square (OPLS) modeling. Based on the results, the groups of compounds that were most influenced by these parameters were determined, and it was concluded that ethanol as the extraction solvent together with low temperatures were the conditions that provided a higher chemical diversity in the extract.