Objective: Oncogenic human papillomaviruses (HPVs) are the etiological agents of cervical cancer. Different cofactors might be needed for malignant transformation, but they still remain elusive. Methods: To delineate the role of Chlamydia trachomatis (CT) and herpes simplex virus type 2 (HSV2) in HPV-positive cervical intraepithelial neoplasia (CIN) lesions and cervical carcinoma a series of 149 cervical cancer and CIN biopsies were analyzed for CT and HSV2 DNA by PCR, and HPV genotyped by InnoLipa. Monitoring of aberrations in key intracellular pathways due to CT/HSV2 and HPV co-expression were analyzed with 13 biomarkers. Results: Of the 149 samples tested, 136 were HPV DNA positive; 32/136 contained also CT DNA and 29 HSV2 DNA. Detection of CT was significantly (p = 0.0001) related to multiple-type HPV infections, while HSV2 was of borderline significance (p = 0.053). Of the 13 biomarkers tested, cytoplasmic and nuclear NF-ĸB and VEGF-C were significantly increased in CT+/HPV+ lesions; p = 0.023, p = 0.045, and p = 0.020 as well as survivin, p = 0.026. Survivin was the only marker that was overexpressed also in HSV2+/HPV+ lesions, p = 0.027. Conclusions: CT infection favors the entry and persistence of multiple HR-HPV types, which leads to viral integration, inhibition of apoptosis, overexpression of E6/E7 oncogenes and cell transformation.