<p>While chemotherapy has traditionally been recognized for its direct cytotoxic effects on cancer cells, increasing attention has been directed towards its capability to modulate tumor immune mechanisms. However, the majority of studies have focused on the quantitative assessment of tumor-infiltrating lymphocytes (TILs), without evaluating their functional status. Therefore, the present study investigated the pre-therapeutic exhaustion status of TILs and its impact on the efficacy of neoadjuvant chemotherapy (NAC) in patients with esophageal squamous cell carcinoma (ESCC). TILs were isolated from 60 endoscopic biopsy samples from ESCC patients before NAC. The proportion of CD8⁺ TILs co-expressing PD-1 and Tim-3 was evaluated using flow cytometry. Their functional status was assessed by examining the expression of exhaustion markers (TOX, LAG-3, CD39), cytotoxic molecules (granzyme B, perforin), and cytokines (IFN-γ, TNF-α). A cytotoxic assay was also performed to evaluate their direct killing capacity against anti-CD3scFv BALL-1 cells. A high proportion of PD-1⁺Tim-3⁺CD8⁺ TILs was significantly associated with a poor pathological response to NAC (<i>p</i> = 0.027) and was identified as an independent predictor of poor response in multivariate analysis (odds ratio = 4.1, 95% confidence interval = 1.1–14.7, <i>p</i> = 0.032). A functional analysis revealed that PD-1⁺Tim-3⁺CD8⁺ TILs expressed high levels of exhaustion markers and low levels of cytotoxic molecules and cytokines and also exhibited impaired cytotoxic function. The proportion of PD-1⁺Tim-3⁺CD8⁺ TILs decreased after NAC in responders. These results suggest the potential of a high proportion of PD-1⁺Tim-3⁺CD8⁺ TILs in the pre-treatment tumor microenvironment as a useful predictor of a poor NAC response in ESCC patients.</p>