The marine environment is an untapped source for biodiversity and has a great potential to provide the drugs of the future. Antibiotic resistance is an increasing threat worldwide and the need for discovering new antibacterial compounds is urgent. Marine microorganisms produce a wide range of bioactive compounds, and marine fungi have only been exploited to a small extent. This creates a great potential for finding novel antibacterial compounds in marine fungi. In this study, the antibacterial and anticancer potential for five marine fungi Acremonium sp. TS7, Typhula sp., Amylocarpus encephaloides, Pseudogymnoascus sp. TS12 and Digitatispora marina have been investigated for antibacterial and anticancer activity. The One-strain-many-compounds (OSMAC)-approach was to try to induce the production of secondary metabolites by applying stress to the marine fungi. These five marine fungi were fermented on four different media and at two different temperatures. Half of the fermentations were co-cultivated with the marine bacteria Leeuwenhoekiella sp. The active fractions were dereplicated with UPLC-QToF-MS. The antibacterial compounds were identified as rhamnolipids and were found in all active samples. Rhamnolipids were also identified in the bacteria controls with Leeuwenhoekiella sp., suggesting that Leeuwenhoekiella sp. is the producer of rhamnolipids. The bioactivity effects of our OSMAC-approach were not as expected, this is probably due to that the culture conditions selected for this study did not trigger the production of secondary metabolites. The full bioactivity potential for Acremonium sp. TS7, Typhula sp., Amylocarpus encephaloides, Pseudogymnoascus sp. TS12 and Digitatispora marina has not been fully investigated in this study, but should be further explored.