PURPOSE: To understand the effect of Nitazoxanide (NTZ), Rapamycin, Thalidomide, alone and in combination with BCG on bladder cancer (BC) histopathology and programmed death-ligand 1 (PD-L1) and anti-cytotoxic T lymphocyte antigen 4 (CTLA4) expression. METHODS: Female Fisher-344 rats underwent intravesical N-methyl-N-nitrosourea (MNU) followed by weekly intravesical treatment with saline (controls, n = 10), BCG (n = 10), NTZ (n = 8), BCG plus NTZ (n = 8), Rapamycin (n = 10) BCG plus Rapamycin (n = 10), Thalidomide (n = 10), and BCG plus Thalidomide (n = 10), and euthanized after 8 weeks and their bladders were investigated for BC and PD-L1 and CTLA4 expression. RESULTS: Rapamicyn alone and in combination with BCG had the lowest number of bladder neoplasias in the histopathology exam (1/10). Neoplastic lesions were found in 4/10 BCG recipients, 5/10 Thalidomide recipients, 4/10 Thalidomide plus BCG recipients, 5/8 NTZ and 3/8 NTZ plus BCG recipients. Adding NTZ to BCG increased the expression of PD-L1 and adding Rapamycin or Thalidomide decreased PD-L1 and CTLA4 expression compared to BCG alone. Rapamycin alone significantly increased CTLA4 and slightly increased PD-L1 expression but its combination with BCG significantly decreased both markers. Thalidomide had a similar effect; however, it was only slightly different from the control and BCG alone groups. CONCLUSION: Intravesical BCG combination treatment seems to effectively prevent BC development in an immunecompetent clinically relevant animal model, introducing Thalidomide, Nitazoxanide, and specially Rapamycin as candidates in the intravesical immunotherapy advancement. Our study contributes in understanding the mechanism of cancer immunotherapy.