Molecular structures, packings, and intermolecular interactions significantly affect the photophysical properties of organic luminogens. In this work, the photoluminescence (PL) and mechanoluminescence (ML) of two pairs of isomers, 1/2 and 3/4, were systematically explored. The fluorescence of crystals 1c and 4c is much brighter than that of their isomers 2c and 3c, respectively. Only 1c is ML-active among all four molecules. Single-crystal structural analysis revealed that isomerization of a substituent group affected their molecular packing and intermolecular interactions. Stronger intermolecular interaction and intact three-dimensional hydrogen-bonded networks were formed only in crystal 1c, which were essential for preventing slippage of molecular layers and generating ML; the other molecules were either lacking π-π interactions or C-H···π interactions. Theoretical calculation suggested that the energy barrier between the Franck-Condon (FC) structure and minimum energy crossing point (MECP) structure of 2/3 was much lower than that of 1/4. Nonradiative decay channels of molecules 2 and 3 were thus more easily activated, which led to their lower quantum yield.