Photodynamic therapy (PDT) is a minimally invasive treatment with low systemic toxicity and immunomodulatory effects, increasingly applied in managing HPV‐associated cervical lesions. Toll‐like receptors (TLRs) are critical in regulating immune responses in cervical pathology, yet their dynamics under PDT remain underexplored. This study investigates the effect of PDT on TLR2, TLR3, TLR4, and TLR8 expression in cervical epithelial cells ex vivo, considering lesion severity. The results reveal that TLR8 exhibited the most significant reduction across all groups 2 h after PDT, with the strongest suppression in patients with invasive cervical cancer. TLR4 expression decreased by 24% in HPV‐infected patients and by 71% in those with cervical cancer, highlighting its potential role in modulating the inflammatory microenvironment post‐PDT. TLR3 showed hyperexpression in LSIL and suppression in HSIL/CIN III, although changes were not statistically significant ( p > 0.05). TLR2 expression remained unchanged, likely due to HPV type variability. These findings demonstrate that PDT effectively reduces hyperexpression of TLR8, TLR4, and TLR3 in early‐stage cervical cancer, suggesting their potential as biomarkers for treatment efficacy. Further research incorporating HPV typing and advanced techniques like liquid biopsy is essential to refine our understanding of TLRs in PDT for HPV‐associated cervical lesions.