Dark septate endophytes (DSEs) are melanin-enriched ascomycetous fungi that are abundant in stressed environments. However, little is known about their physiology and metabolism, and DSE genes have not been functionally characterized yet. Therefore developing molecular genetic tools to investigate the biological function of genes of interest in DSEs is of major significance. We investigated Agrobacterium tumefaciens-mediated transformation (ATMT) efficiency in eight DSE strains belonging to Cadophora sp., Cadophora malorum, Leptodontidium sp., Phialophora mustea, and Cladosporium cladosporioides. ATMT efficiency was DSE-dependent and ranged from 0.6 to 125 %. We further focused on the effect of mycelium ageing on ATMT efficiency. Leptodontidium sp. Me07, Leptodontidium sp. Pr30, and C. cladosporioides CBS 101367 were significantly more transformed using 15-d-old mycelium (44.5, 6.9, and 1.1 %, respectively) as compared to 2-d-old mycelium (121, 28.7, and 25.1, respectively), whereas P. mustea Pr29 was more transformed using young mycelium (21.5 % compared to 5.3 % for the old mycelium). Finally, we focused on the effect of melanin content on ATMT efficiency. Melanin content in mycelium ranged from 0.9 to 3.2 mg g DW. Tricyclazole negatively modulated melanin content, while copper positively modulated it. However there was no correlation between hyphal melanin content and ATMT efficiency.