Stereotactic needle biopsy (SNB) may be performed to collect tissue samples from lesions not amenable to open surgery. Integration of tractography, intraoperative imaging and fluorescence has been applied to reduce risk of complications and confirm the adequacy of bioptic specimens. Clinical and radiological data from patients who underwent stereotactic needle biopsy with the use of intraoperative CT, tractography and 5-aminolevulinic acid (5-ALA) fluorescence in a single Hospital were retrospectively reviewed to evaluate the accuracy and safety of the procedure. Seven patients were included in the study, and all the collected specimens showed red fluorescence. In six of them, the final histopathological diagnosis was grade 4 glioblastoma IDH-wt and in the other case it was Diffuse large B-Cell Lymphoma. The integration of tractography, intraoperative CT and 5-ALA as an intraoperative marker of diagnostic samples may be suggested in biopsies of suspect gliomas and lymphomas. The cost-effectiveness of the procedure should be evaluated in future studies.