Background Overexpression of viral oncogenes E6 and E7 of high-risk (HR)–human papillomavirus (HPV) plays a key role in the progression to cervical intraepithelial neoplasia (CIN) and cancer. Detection of E6/E7 mRNA may help distinguish between transient and transforming infections, thus improving cervical cancer screening accuracy. This study aimed to evaluate the association of E6/E7 mRNA expression with HR-HPV infection, cervical cytology, and histologically confirmed cervical lesions and to compare its agreement with HPV DNA testing and cytology. Methods: A total of 526 women undergoing cervical cancer screening between January 2023 and May 2025 were enrolled. E6/E7 mRNA expression was assessed using flow cytometry with the HPV OncoTect 3Dx E6/E7 mRNA assay and compared with HR-HPV DNA testing, cervical cytology, and available histopathologic findings from biopsy specimens. Results: Among 526 samples, eight (1.5%) tested positive for E6/E7 mRNA, compared to 30 (5.7%) HR-HPV DNA–positive and 17 (3.2%) abnormal cytology results. Median E6/E7 mRNA expression was higher in HR-HPV– positive cases and in cytologically abnormal cases. Almost perfect agreement was observed between E6/E7 mRNA results, HR-HPV DNA testing, and cytology. Histopathologic follow-up was available for nine women with cytological abnormalities ≥low-grade squamous intraepithelial lesion, including four CIN2 and five CIN1 cases. E6/E7 mRNA expression was markedly elevated in one CIN2 case associated with HPV16 infection (37.51%), while two CIN1 cases demonstrated high mRNA expression despite negative HPV DNA results. Conclusions: Flow-cytometric E6/E7 mRNA detection showed high overall agreement with HPV DNA testing and cytology in this screening cohort. Larger prospective studies with comprehensive histologic follow-up are needed to determine its clinical utility.