Surgical resection is the primary treatment for malignant brain tumors. This procedure has a dilemma—aggressive surgical resection tends to extend patient survival; however, it also increases the risk of neurological deficiencies. Current medical imaging methods are not sensitive and their interpretation largely depend on surgeon’s impression. High-speed cellular imaging method by using clinically applicable moxifloxacin was demonstrated for fast and sensitive tumor-detection. The detailed cytoarchitecture of brain tumor mouse model and malignant human brain tumors was revealed. This study showed the potential and feasibility of moxifloxacin-based confocal microscopy as a surgery-guiding method for tumor removal.