Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera Cadophora sp., Leptodontidium sp. and Phialophora mustea, were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of Leptodontidium sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of Leptodontidium albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.