Deep-sea fungi have evolved extreme environmental adaptation and possess huge biosynthetic potential of bioactive compounds. However, not much is known about the biosynthesis and regulation of secondary metabolites of deep-sea fungi under extreme environments. Here, we presented the isolation of 15 individual fungal strains from the sediments of the Mariana Trench, which were identified by internal transcribed spacer (ITS) sequence analysis as belonging to 8 different fungal species. High hydrostatic pressure (HHP) assays were performed to identify the piezo-tolerance of the hadal fungi. Among these fungi,Aspergillus sydowiiSYX6 was selected as the representative due to the excellent tolerance of HHP and biosynthetic potential of antimicrobial compounds. Vegetative growth and sporulation ofA. sydowiiSYX6 were affected by HHP. Natural product analysis with different pressure conditions was also performed. Based on bioactivity-guided fractionation, diorcinol was purified and characterized as the bioactive compound, showing significant antimicrobial and antitumor activity. The core functional gene associated with the biosynthetic gene cluster (BGC) of diorcinol was identified inA. sydowiiSYX6, named asAspksD. The expression ofAspksDwas apparently regulated by the HHP treatment, correlated with the regulation of diorcinol production. Based on the effect of the HHP tested here, high pressure affected the fungal development and metabolite production, as well as the expression level of biosynthetic genes which revealed the adaptive relationship between the metabolic pathway and the high-pressure environment at the molecular level.