The constant need to search for new drugs is a major driver for the discovery of new molecules of pharmaceutical interest. Natural products (NPs) of microbial origin have been recognized for their therapeutic properties, with Actinomycetota being one of the leading groups in terms of their production. Due to the fact that Actinomycetota contain in their genomes a high number of biosynthetic gene clusters that may not be expressed under common cultures conditions, the strategy known as “one strain many compounds” (OSMAC) has emerged as an important approach to expand the chemical diversity of actinobacterial metabolites. In this work, 8 OSMAC conditions were applied to 10 actinobacterial isolates previously obtained from deep-sea samples collected at Madeira and Azores archipelagos, Portugal, in an attempt to activate silent biosynthetic gene clusters capable of producing new NPs. Organic extracts from the isolates grown under the different conditions (80 in total) were tested for their antimicrobial, anticancer and anti-inflammatory activities, revealing 11 extracts that inhibited the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhimurium or Candida albicans, and 9 extracts that reduced the cellular viability of T-47D or HepG2 cancer cells, while no anti-inflammatory activity was observed. Metabolomic profile of the actinobacterial extracts revealed metabolites matching known NPs, as well as features suggestive of previously unreported compounds (15 in total). This study demonstrated that the OSMAC approach is effective in modulating secondary metabolism in Actinomycetota and is consequently a useful resource for the discovery of new molecules with biotechnological potential.