In clinical practice, imiquimod is used to treat Human Papillomavirus (HPV)-related lesions, such as condyloma and Cervical Intraepithelial Neoplasia (CIN). Metronidazole is the most commonly prescribed antibiotic for bacterial vaginosis. The study developed biodegradable imiquimod- and metronidazole-loaded nanofibrous mats and assessed their effectiveness for the topical treatment of cervical cancer, a type of HPV-related lesion. Nanofibers of two distinct poly[(d,l)-lactide- The experimental data show that drug-eluting nanofibers could discharge in vitro high concentrations of imiquimod and metronidazole for exceeding 30 days. In vivo, each membrane consistently released elevated concentrations of imiquimod/metronidazole at the intended site in mice over a four-week period, with minimal systemic drug concentration detected in the bloodstream. The mice treated with drug-loaded nanofibers displayed noticeably reduced tumor volumes compared to both the control group and the group treated with pristine nanofibers. Histological examination revealed the absence of any discernible tissue inflammation. Biodegradable nanofibers with a sustainable release of imiquimod and metronidazole demonstrated their effectiveness and lasting impact of treating mice with cervical cancer.