Aims Identification of the mycobiota associated to the marine echinoderm Holothuria poli and investigation of cytotoxic and pro-osteogenic potential of isolated strains. Methods and results Fungal strains were isolated from the animal's body-wall, intestine and faeces. The species identification was based on DNA barcoding and morphophysiological observations. Forty-seven species were identified, all are Ascomycota and mainly belonging to Aspergillus and Penicillium genera. Sixteen strains were grown on three media for chemical extraction. Cytotoxic activity was tested on a hepatic cancer cell line (HepG2), the cells viability was evaluated after treatment using a resazurin based assay (AlamarBlue). Pro-osteogenic activity was tested on human Mesenchymal stem cell, differentiation was measured as the alkaline phosphatase production through reaction with p-nitrophenylphosphate or as the cells ability to mineralize calcium using a colorimetric kit (StanBio). Cytotoxic activity was recorded for four fungal species while five of 48 extracts highlighted bioactivity towards human mesenchymal stem cells. Conclusions The presence of relevant animal-associated mycobiota was observed in H. poli and selected strains showed cytotoxic potential and pro-osteogenic activity. Significance and Impact of the Study Our work represents the first report of a Mediterranean Sea cucumber mycobiota and highlights the isolates potential to synthetize compounds of pharmaceutical interest for regenerative medicine.