Isoindigo (IID) is widely used as a building block for the fabrication of organic semiconductor devices. Understanding the impact of cross-conjugation and linear conjugation on the optoelectronic properties of disubstituted IID is of great importance for the design of improved materials. In this study, phenyl and thienyl groups were substituted at the cross-conjugated 7,7′ position of IID to generate three novel organic semiconductor structures with a donor–acceptor architecture. The optoelectronic properties of this IID derivative were investigated and compared with those of the 6,6′ linearly conjugated IID analogs using UV–Vis spectroscopy and cyclic voltammetry. The experimental results were compared using density functional theory calculations to provide structure–property relationships based on substitution types and attachment sites for IID. The frontier orbital energy levels of the material did not vary dramatically with the position of the substituent, while the type of substituent showed a more significant influence on the HOMO’s energy level and oscillator strength. Phenyl-disubstituted 7,7′ IID (7Ph7′Ph) and thienyl-disubstituted 7,7′ IID (7Th7′Th) materials were used as electron transport layers in perovskite solar cells with a power conversion efficiency of 5.70% and 6.07%, respectively. These observations enhance our understanding of the electronic structure and optoelectronic properties of IID, guiding the design of the next generation of IID-based semiconductors.