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% trans isomer, which upon irradiation with a 365 nm light, isomerized to 81% cis isomer, that showed t (1/2) of >38 h at 37 °C. Back-irradiation of the cis-enriched PSS with a 475 nm light yielded 79% trans-enriched PSS. Interestingly, cis- and trans-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 trans-enriched PSS of 2 (1.2 times less activity). In the 2D cell culture study, the cis-enriched PSS of 2 (IC(50) of 4.8 ± 0.5 μM) was found to be 1.7-fold more potent than its trans-enriched PSS. Notably, the 3D cell spheroid culture study displayed a better photopharmacological response, in which the cis-enriched PSS induced 2.5-fold higher spheroid growth inhibition than the trans-enriched PSS. Cell cycle analysis demonstrated the arrest of the cell cycle in the G1 phase, which occurred more efficiently using the cis isomer than the trans isomer. An immunoblot assay confirmed the inhibition of Stat3 activation. These experimental results agreed with the in silico docking performed on the SH2 domain of Stat3.