Antibiotic resistance is a big threat to global health, as the treatment of a growing number of bacterial infections is becoming a serious problem. In addition, cancer is responsible for the death of millions of people every year and resistance to available therapies is of big concern. It is well established that Actinobacteria are prolific producers of bioactive compounds with pharmaceutical applications. Bioprospecting unexplored or underexplored environments, like the deepsea, may be a key for the discovery of new bioactive molecules. This work aimed to study the biodiversity of the cultivable Actinobacteria associated with deep-sea samples from Portugal, and investigate the antimicrobial and cytotoxic potential of the isolated strains. Nine deep-sea samples, that included sponges, corals and sediments, were collected at the Madeira archipelago at depths between 463 m and 865 m, using the submersible Lula1000. A heat pre-treatment and three selective culture media supplemented with different antibiotics were used to promote the selection of Actinobacteria. Sixty-eight actinobacterial strains were isolated from the analyzed samples, being affiliated with the genera Brevibacterium, Tsukamurella, Microbacterium, Micrococcus, Leucobacter, Rhodococcus, Brachybacterium and Streptomyces. Two of these actinobacterial isolates may represent new Microbacterium species as their 16S rRNA gene similarity was below the cut-off value of 98.7%, used to discriminate between species. The crude extracts of the isolated actinobacterial strains were screened for antimicrobial and cytotoxic activities, using the disk diffusion method and MTT assay, respectively. Two actinobacterial strains associated with the genera Brevibacterium and Brachybacterium were active against one or more of the reference strains tested, namely Candida albicans, Bacillus subtilis and Staphylococcus aureus, exhibiting MIC values in the range of 1000 μg mL-1. Cytotoxic assays revealed 23 strains capable of reducing the cellular viability of at least one of the cell lines tested (T47-D, HepG2 and hCMEC/D3). This work contributed to increase the knowledge about the diversity of Actinobacteria associated with deep-sea samples of Portuguese environments and of their bioactive potential. In the future, dereplication of the bioactive extracts will be performed to look for the presence of new bioactive compounds.