Background: There is an urgent need for novel treatment options for patients with glioblastoma, the most frequent malignant primary brain tumor. In contrast to other types of cancer, immunotherapeutic approaches have so far not been successful against glioblastoma. Converting the glioma microenvironment from a "cold" and immunosuppressive status into a more "hot" and immunopermissive phenotype may allow for clinically meaningful anti-tumor immune responses. Methods: We explored the activity of novel immunocytokines based on the L19 antibody, specific to a tumor-associated epitope of extracellular fibronectin, for the targeted delivery of three pro-inflammatory cytokines (IL-2, IL-12, TNF) to the microenvironment of gliomas. Following an extensive preclinical assessment in 2 orthotopic immunocompetent mouse glioma models, we used a fully-human L19-hTNF fusion protein to treat human patients with recurrent glioblastoma. Results: Intravenous administration of L19-mIL12 or L19-mTNF prolonged survival and cured a proportion of tumor-bearing mice while no effect was seen with L19-IL2. When L19-mIL12 or L19-mTNF were administered to glioma-bearing RAG^−/− mice, no therapeutic activity was observed which suggests adaptive immunity as an underlying mechanism. On a mechanistic level, both immunocytokines induced the infiltration of the tumor site with lymphocytes and promoted the expression of pro-inflammatory cytokines in the tumor microenvironment. In addition, L19-mTNF induced tumor necrosis. Based on these preclinical findings, we initiated a phase I/II clinical trial with a fully-human L19-hTNF fusion protein for patients with isocitrate dehydrogenase (IDH1R132H) wildtype WHO Grade III or IV glioma at first relapse (NCT03779230). Treatment was safe and well tolerated in the first three glioblastoma patients. Administration of L19-hTNF resulted in reduced regional blood perfusion in the tumor region and was associated with more necrotic areas within the tumor as well as an increased number of tumor-infiltrating CD4 and CD8 T cells. Conclusions: The data obtained with the comprehensive preclinical characterization and subsequent clinical translation form the basis for future studies with immunocytokines as novel treatment option for patients with malignant brain tumors. Clinical trial information: NCT03779230.