Intravesical treatment with bacillus Calmette‑Guerin (BCG) is the most common treatment for preventing progression and recurrence of non‑muscle invasive bladder cancer. Our previous study using the N‑butyl‑N‑(4‑hydroxybutyl) nitrosamine (BBN)‑induced orthotopic bladder cancer model demonstrated that intravesical treatment with mitomycin C (MMC) and adriamycin (ADM) suppressed pro‑tumoral immunity, including the aggregation of tumor‑associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. Previous evidence supports the association of resistance to intravesical treatment of BCG with TAMs and Tregs. In the present study, we investigated the antitumoral efficacy of sequential intravesical treatments with chemotherapeutic agents and BCG in a BBN‑induced orthotopic bladder cancer model. Thirty‑six C57BL/6J mice bearing bladder cancer were randomly divided into six treatment groups as follows: control, BCG, MMC, ADM, MMC‑BCG and ADM‑BCG. Intravesical treatment was performed once a week for six weeks. One week after the completion of intravesical treatment, bladder and blood were harvested. MMC‑BCG and ADM‑BCG were more effective antitumor activities than BCG monotherapy. Bladders were subjected to immunohistochemical analysis and revealed that intravesical BCG treatment combined with MMC/ADM promoted the local recruitment of NK cells to the bladder as effectively as BCG monotherapy and reduced TAMs and Tregs in the bladder. Interleukin (IL)‑17 and granulocyte‑colony stimulating factor (G‑CSF) in serum were analyzed by enzyme‑linked immunosorbent assay and these levels were revealed to be elevated in mice treated with sequential treatments similar to levels following monotherapy with MMC and ADM. Our findings indicated that intravesical sequential treatment could suppress the resistance to BCG through the enhancement of antitumor immunity (induction of NK cells) and inhibition of pro‑tumoral immunity (reduction of TAMs and Tregs). Systemic changes in IL‑17 and G‑CSF may be involved in topical immunomodulation. Further studies including clinical trials may be required to establish an appropriate strategy based on the immunomodulation of the tumor microenvironment.