For bladder cancer, intravesical chemo/immunotherapy is widely used as adjuvant therapy after surgical transurethral resection. Bacillus Calmette-Guerin (BOG) is a live attenuated Mycobacterium of the same family as tuberculosis, that is capable of inducing a local inflammatory response upon instillation into the bladder. Intravesical therapy with BCG has proved to be more effective in the prophylaxis and treatment of superficial bladder tumors than most chemotherapeutic agents used for the same indication. However, compared to intravesical chemotherapy, BCG immunotherapy provokes more pronounced local and systemic reactions. In addition to the commonly induced granulomatous inflammatory changes in the bladder, which produce irritative symptoms, this therapy may cause systemic side effects varying from mild malaise and fever to, in rare instances, life-threatening or fatal sepsis. Nanoparticles with positive surface charge and mucoadhesive properties were developed to overcome these side effects. Hence, the aim of this study was to optimize and evaluate cationic chitosan (CS) nanoparticles encapsulating BCG in terms of antitumor efficacy after intravesical administration in bladder tumor, induced in rat model. It was found that nanoparticle formulations of 269-375 nm in size can be produced with 42% encapsulation efficiency. The zeta potential was positive and was suitable for intravesical administration. Antitumor efficacy was determined over the parameters of histopathological evaluation, survival rate and mean bladder weight in comparison to treatment with commercial BCG solution. Concerning survival rates, BOG-loaded chitosan nanoparticles resulted in significantly longer survival than BCG commercial product (up to 86 days of survival with no systemic side effects). When compared to healthy bladder weight averages, all groups (especially BCG commercial solution) showed higher bladder weights confirming tumor formation. Histopathological findings confirmed antitumor activity in all treatment groups and optimum findings were observed in groups treated with CS nanoparticles encapsulating BCG. At the same time, significant nanoparticle accumulation in bladder tissues was observed especially for BCG-loaded CS group. In this study, it was clearly observed that cationic CS nanoparticles provide a significantly improved perspective in intravesical immunotherapy of bladder tumors.