Patients with non‑muscle invasive bladder cancer (NMIBC) frequently relapse following surgery due to incomplete resection and chemoresistance, highlighting the importance of developing novel therapeutic strategies that mechanistically assist in eradicating the residual tumor. The aim of the present study was to evaluate the anticancer effect of chlorophyllin e6‑mediated photodynamic therapy (e6‑PDT) and its potential mechanisms by using monolayer cells or multicellular tumor spheroid models of human bladder cancer cells (T24 and 5637). The results revealed that e6‑PDT exhibited significant cytotoxicity in the T24 and 5637 cells of these two models as detected by the Water‑Soluble Tetrazolium Salts‑1 and CellTiter‑Glo Luminescent Cell Viability assays, respectively. Cell migration and invasion capacities decreased markedly following e6‑PDT. In addition, the cells following e6‑PDT exhibited typical morphological changes of apoptosis as detected by fluorescence microscopy with 4',6‑diamidino‑2‑phenylindole staining and transmission electron microscopy. A greater number of apoptotic cells were observed post‑e6‑PDT by flow cytometry. The expression levels of poly(adenosine diphosphate‑ribose) polymerase (PARP) and B‑cell lymphoma 2 protein were decreased, while cleaved PARP was increased, significantly following e6‑PDT as determined by western blotting. The level of intracellular reactive oxygen species (ROS) was increased, while the activity of superoxide dismutase (SOD) was decreased, significantly in e6‑PDT‑treated cells. Thus, the novel e6‑PDT exhibits prominent photo‑cytotoxicity effect and the induction of apoptosis was probably due to the inhibition of SOD activity and the generation of ROS. These results indicate that chlorophyllin e6 is an effective photosensitizer and that e6‑PDT may have a therapeutic application for the treatment of bladder cancer.