Video-rate structured illumination microscopy (VR-SIM) offers a potential alternative to destructive pathology at the point of procedure in prostate biopsy. Sensitivity of VR-SIM ranged from 63–88%, improving to 75–92% when biopsies had >5% tumour content, and specificity ranged from 78–89%. VR-SIM offers good image quality and is fast and easy to perform, which are favourable features for point-of-procedure biopsy pathology.