Oceans cover >70% of the earth and encompass variable habitats concerning salinity, temperature, pressure, light availability. The deep sea (>1000 m water depth) constitutes more than 60% of the ocean´s biosphere and harbors an unparalleled biodiversity. It constitutes an extreme habitat due to high pressure, darkness and often low nutrient and oxygen concentrations. In order to ensure their survival, microorganisms thriving in such environments have to develop unique metabolic adaptations, thus represent an interesting resource for the discovery of new molecules. However, due to access difficulties to deep-sea habitats and the lack of suitable and affordable sampling techniques, deep-sea microorganisms have remained untapped for their potential in marine biodiscovery. In this study, we obtained deep-sea sediment samples from Arctic Ocean (-2432 m), sampled by an ROV during RV Polarstern expedition 108. Isolation of microorganisms has been performed using two specific media for bacteria and fungi, respectively. Isolates were identified by amplification of the 16S rRNA gene (bacteria) and ITS1-2 region (fungi) followed by Sanger sequencing. In total, 70 bacterial isolates were identified covering four phyla (52 Firmicutes, 1 Actinobacteria, 11 Proteobacteria and 6 Bacteroidetes) and seven fungal strains from two different phyla (6 Ascomycota and 1 Basidiomycota). Selected isolates were cultivated in two different media, followed by solvent (EtOAc) extraction and bioactivity screenings against a panel of clinically relevant microbial pathogens and six cancer cell lines. At 100 µg/mL concentration, three bacterial extracts showed antitumor activity (>70%), whereas 17 exhibited activity (>65%) against methicillin-resistant Staphylococcus aureus (MRSA). Notably, only one fungus showed a cultivation medium dependent-high antifungal activity (>90%), highlighting the impact of culture media on the production of bioactive secondary metabolites.