Photodynamic therapy (PDT) utilizes optical energy to activate a pre-administered photosensitizer drug to achieve a localized tumor control. In the presented study, PDT mediated with a second-generation photosensitizer, WST09 (TOOKAD, Steba Biotech, The Netherlands), is investigated as an alternative therapy in the treatment of prostate cancer. In vivo canine prostate is used as the animal model. PDT was performed by irradiating the surgically exposed prostates both superficially and interstitially with a diode laser (763 nm) to activate the intra-operatively i.v. infused photosensitizer. During light irradiation, tissue optical properties, and temperature were monitored. During the one-week to 3-month period post PDT treatment, the dogs recovered well with little or no complications. The prostates were harvested and subjected to histopathological evaluations. Maximum lesion size of over 3 cm in dimension could be achieved with a single treatment, suggesting the therapy is extremely effective in destroying prostatic tissue. Although we found there was loss of epithelial lining in prostatic urethra, there was no evidence it had caused urinary tract side effects as reported in those studies utilizing transurethral irradiation. In conclusion, we found second generation photosensitizer WST09 mediated PDT may provide an excellent alternative to treat prostate cancer.