Stat3 protein is known to be hyperactive in many human cancer cells, including leukaemia, lymphoma, breast, ovarian, lung, and prostate cancer cells. The treatment of most of these cancer types relies on chemotherapy, which encounters various side effects owing to off-target effects of the drugs. Photopharmacology promises to eliminate such off-target effects with the use of photoswitchable drugs. Based on a potent Stat3 inhibitor, in this study, we developed two azobenzene-based photoswitchable inhibitors 2 and 3. Ambient light-exposed compound 2 contained 90% isomer, which upon irradiation with a 365 nm light, isomerized to 81% isomer, that showed of >38 h at 37 °C. Back-irradiation of the -enriched PSS with a 475 nm light yielded 79% -enriched PSS. Interestingly, - and -enriched PSSs showed 1.3-1.5 times higher anticancer potencies against the MDA-MB-231 breast cancer cell line than the parent Stat3 inhibitor 1, except the -enriched PSS of 2 (1.2 times less activity). In the 2D cell culture study, the -enriched PSS of 2 (IC of 4.8 ± 0.5 μM) was found to be 1.7-fold more potent than its -enriched PSS. Notably, the 3D cell spheroid culture study displayed a better photopharmacological response, in which the -enriched PSS induced 2.5-fold higher spheroid growth inhibition than the -enriched PSS. Cell cycle analysis demonstrated the arrest of the cell cycle in the G1 phase, which occurred more efficiently using the isomer than the isomer. An immunoblot assay confirmed the inhibition of Stat3 activation. These experimental results agreed with the docking performed on the SH2 domain of Stat3.