It is an intermediate in the production of the biological pigment eumelanin.

[2] 5,6-Dihydroxyindole is biosynthesized from L-dopachrome in a reaction catalyzed by a tyrosinase enzyme and is further converted into indole-5,6-quinone.

[3] In humans, 5,6-dihydroxyindole is involved in the metabolic disorder hawkinsinuria.

[3] In some insects, 5,6-dihydroxyindole is a reactive compound that is produced as a component of defense responses and has antibacterial and antifungal activity.

[5] This compound is condensed with nitromethane in a Henry reaction, followed by nitration, reduction of the nitro groups, and hydrogenolysis of the benzyl protecting groups.