Porphodilactones represent the porphyrin analogues, in which the peripheral bonds of two pyrrole rings are replaced by lactone moieties. They provide an opportunity to investigate how β-substituent orientation of porphyrinoids modulates the electronic structures and optical properties, in a manner similar to what is observed with naturally occurring chlorophylls. In this work, a comprehensive description of the synthesis, characterization, and optical properties of meso-tetrakispentafluorophenylporphodilactone isomers is first reported. The β-dilactone moieties are found to lie at opposite pyrrole positions (trans- and cis-configurations are defined by the relative orientations of the carbonyl group when one lactone moiety is fixed), in accordance with earlier computational predictions (Gouterman, M. J. Am. Chem. Soc. 1989, 111, 3702). The relative orientation of the β-dilactone moieties has a significant influence on the electronic structures and photophysical properties. For example, the Qy band of trans-porphodilactone is red-shifted by 19 nm relative to that of the cis-isomer, and there is a 2-fold increase in the absorption intensity, which resembles the similar trends that have been reported for natural chlorophyll f and d. An in depth analysis of magnetic circular dichroism spectral data and TD-DFT calculations at the B3LYP/6-31G(d) level of theory demonstrates that the trans- and cis-orientations of the dilactone moieties have a significant effect on the relative energies of the frontier π-molecular orbitals. Importantly, the biological behaviors of the isomers reveal their different photocytotoxicity in NIR region (>650 nm). The influence of the relative orientation of the β-substituents on the optical properties in this context provides new insights into the electronic structures of porphyrinoids which could prove useful during the development of near-infrared absorbing photosensitizers.