Background Early diagnosis of non-melanoma skin cancer (NMSC) is potentially possible using optical coherence tomography (OCT) which provides non-invasive, real-time images of skin with micrometre resolution and an imaging depth of up to 2 mm. OCT technology for skin imaging has undergone significant developments, improving image quality substantially. The diagnostic accuracy of any method is influenced by continuous technological development making it necessary to regularly re-evaluate methods. Objective The objective of this study is to estimate the diagnostic accuracy of OCT in basal cell carcinomas (BCC) and actinic keratosis (AK) as well as differentiating these lesions from normal skin. Methods A study set consisting of 142 OCT images meeting selection criterea for image quality and diagnosis of AK, BCC and normal skin was presented uniformly to two groups of blinded observers: 5 dermatologists experienced in OCT-image interpretation and 5 dermatologists with no experience in OCT. During the presentation of the study set the observers filled out a standardized questionnaire regarding the OCT diagnosis. Images were captured using a commercially available OCT machine (Vivosight ®, Michelson Diagnostics, UK). Results Skilled OCT observers were able to diagnose BCC lesions with a sensitivity of 86% to 95% and a specificity of 81% to 98%. Skilled observers with at least one year of OCT-experience showed an overall higher diagnostic accuracy compared to inexperienced observers. Conclusions The study shows an improved diagnostic accuracy of OCT in differentiating AK and BCC from healthy skin using state-of-the-art technology compared to earlier OCT technology, especially concerning BCC diagnosis.