Unsubstituted 2,5-bis(2,2′-bithiophene-5-yl)-1,3,4-thiadiazole and four of its derivatives containing solubilizing octyl groups in different positions of the terminal thiophene ring were synthesized (3 new compounds and 2 already reported). Their UV–vis absorption and emission spectra turned out to be strongly dependent on the position of the substituent and showed significant bathochromic shifts of the dominant transition for compounds with the substituents attached to Cα of the terminal ring (λmax > 425 nm). A good correlation was found for the experimentally determined and theoretically calculated excitation energies employing the time-dependent Coulomb-attenuated hybrid exchange-correlation functional using the 6-31G* basis set (TD CAM-B3LYP/6-31G*). The calculations showed, in addition, that the alkyl substituents improved the planarity of the molecule and its aromaticity, and that they raised the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) levels (the latter to a lesser extent) via electron-donating effects. The only exception was the compound with the substituent attached to the inner Cβ position. In this case, the absorption band originating from the dominant transition was hypsochromically shifted in comparison to the corresponding transition in the unsubstituted compound, again in perfect agreement with the quantum chemical calculations, which predicted a less planar structure of this derivative. Theoretical calculations concerning the substituent-induced shifts of the HOMO and LUMO levels were found to be in good accordance with the ionization potential and the electron affinity changes determined by cyclic voltammetry, which showed the same trend. Thin films of the studied semiconductors deposited on SiO2/Si showed a highly anisotropic structure with molecules stacked almost perpendicular to the surface. Among all derivatives studied, the two compounds with substituents attached to the Cβ position formed ordered monolayers on highly oriented pyrolytic graphite in which molecular packing was much less dense than in 3D crystals. Preliminary investigations demonstrated that the synthesized new semiconductors could be used for fabrication of efficient light-emitting diodes.