Background: Pleural biopsies in patients with suspected malignant pleural mesothelioma (MPM) are often inconclusive resulting in repeat diagnostic procedures. Confocal laser endomicroscopy (CLE) enables real-time imaging on a cellular level. We investigated pleural CLE imaging as a biopsy guidance technique to distinguish malignant from benign pleural disease. Methods: Prospective, multicenter study in patients with (suspected) MPM based on PET-CT imaging who were scheduled for pleural biopsies. Patients received 2.5 mL fluorescein intravenously preceding the procedure. In vivo through-the-needle CLE imaging of the pleura and ex vivo CLE imaging of the biopsies were correlated with histology. CLE characteristics for various pleural entities were identified, and their interpretability was tested by CLE video scoring by multiple blinded raters. Results: CLE imaging was successfully obtained in 19 of 20 diagnostic pleural biopsy procedures (thoracoscopy: n = 4, surgical excision: n = 3, CT scan: n = 3, ultrasound: n = 9, esophageal ultrasound guided: n = 1) in 15 patients. CLE videos (n = 89) and corresponding pleural biopsies (n = 105) were obtained. No study-related adverse events occurred. Tumor deposits of MPM were distinguished from areas with pleural fibrosis based on CLE imaging and recognized by raters (n = 3) (interobserver agreement, 0.56; 95% CI, 0.49-0.64). Conclusions: CLE imaging was feasible and safe regardless of the biopsy method. Real-time visualization of pleural abnormalities in epithelial and sarcomatoid MPM could be distinguished from pleural fibrosis. Therefore, CLE has potential as a guidance biopsy tool to reduce the current substantial rate of repeat biopsy procedures by identification of areas with malignant cells in vivo (smart needle). Trial Registry: ClinicalTrials.gov; No.: NCT02689050; URL: www.clinicaltrials.gov.