The rising of pathological processes was proved to induce alterations of the intrinsic autofluorescence properties of biological tissues, that can be exploited for diagnostic purposes. In oncology, applications of autofluorescence as a parameter for in situ cancer detection in several organs are reported. In this work, autofluorescence properties of normal and tumor tissue in the brain are described, suitable for a real-time diagnostic application. Studies were performed both on ex vivo resected samples, by microspectrofluorometric techniques, and in vivo, during surgical operation, by means of fiber-optic probe. Significant differences were found in autofluorescence emission properties between normal and tumor tissues, concerning both spectral shape, peak position and signal amplitude. The potential of autofluorescence as a parameter to distinguish neoplastic from normal condition opens interesting prospects for improving of the efficacy of neurosurgical operations, by allowing an intraoperative delineation of tumor resection margins through a noninvasive technique.