Lesion biopsy is currently used to diagnose erythroplasia of Queyrat (EQ), a rare squamous cell carcinoma in situ of the glans penis, or to determine whether the cancer is invasive, although the results only apply to the area from which the biopsy is taken. In this case report, we illustrate for the first time the use of optical coherence tomography (OCT) in imaging the entire lesion in a patient with EQ. The results confirmed that the patient had in situ rather than invasive carcinoma. Consequently, non-invasive treatment with imiquimod 5%, a topical immunomodulator with antitumour and antiviral properties, was initiated. Excellent clinical results were observed 4 weeks after the patient had been treated with imiquimod 5% three times a week for 8 weeks, which were confirmed using OCT imaging. One year later, there was still no evidence of pathology either clinically or via OCT imaging. OCT imaging should be used in conjunction with biopsy evaluation in the diagnostic work-up of EQ. Imiquimod 5% is a suitable treatment for patients with EQ, and the treatment response can be evaluated using OCT.