A series of nine novel indigo derivatives, including diiodoindigo, octahalogenated indigoids and compounds with extended π-conjugated system, were synthesized, characterized and investigated as semiconductor materials in organic field-effect transistors (OFETs). Among them, 6,6′-diiodoindigo demonstrated the ambipolar behavior with balanced p-type and n-type mobilities. The complete substitution of hydrogens at the indigo core with halogen atoms led to low electron mobilities in OFETs. An extension of the conjugated system through the introduction of small aromatic substituents (thiophene and phenyl) resulted in predominant p-type behavior. Fusion of aromatic rings resulted in z-shaped dibenzoindigo, which showed poor charge transport properties due to the non-optimal arrangement of molecules along each other in the crystal lattice. The acquired data fulfilled the previously reported model based on the relationship between the chemical nature of substituents and their positions at the indigo core, optoelectronic properties of materials and their performance in OFETs. The results of this study will be useful for rational design of a new generation of the indigo-based semiconductors for biocompatible organic electronics.