Background The current study aimed to investigate the dynamic alteration and prognostic significance of tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and PD-L1 status of immune cells in muscle-invasive bladder cancer (MIBC) treated with neoadjuvant chemotherapy (NAC). Methods Multiplex immunofluorescence staining was performed to examine CD68(+) TAM, CD4(+) T cell, CD8(+) T cell, FOXP3(+) Treg cell, and PD-L1 expression in paired MIBC tissues (n = 54) before and after NAC. Patients were then divided into definite responders (DR), (<= pT1) and incomplete responders (IR). Results There was no significant difference between DR and IR cohorts for the immune cell infiltration levels at the baseline status. Tobacco history was identified to be associated with worse NAC efficacy. CD68(+) (stroma area: p = 0.025; tumor area: p = 0.028; total area: p = 0.013) and CD68(+)PD-L1(-) (stroma area: p = 0.035; tumor area: p = 0.013 total area: p = 0.014) TAMs infiltration levels decreased significantly after NAC, while there was no significant difference of CD68(+)PD-L1(+) and TILs. The infiltration of CD68(+) (p = 0.033), CD68(+)PD-L1(-) (p = 0.033), and CD68(+)PD-L1(+) (p < 0.001) TAMs in stroma area were significantly associated with poorer disease-free survival rate (DFS) of MIBC patients. Conclusion CD68(+) and CD68(+)PD-L1(-) TAMs infiltration levels decreased significantly after NAC and pre-treatment TAM infiltration levels were independent prognostic factors for MIBC patients. While there was no sufficient evidence demonstrating that pre-treatment TILs or TAMs could predict response to NAC in MIBC patients.