Introduction: Incomplete head and neck cancer resection occurs in up to 85% of cases, leading to increased odds of local recurrence and regional metastases; thus, image-guided surgical tools for accurate, in situ and fast detection of positive margins during head and neck cancer resection surgery are urgently needed. Oral epithelial dysplasia and cancer development is accompanied by morphological, biochemical, and metabolic tissue and cellular alterations that can modulate the autofluorescence properties of the oral epithelial tissue. Objective: This study aimed to test the hypothesis that autofluorescence biomarkers of oral precancer and cancer can be clinically imaged and quantified by means of multispectral fluorescence lifetime imaging (FLIM) endoscopy. Methods: Multispectral autofluorescence lifetime images of precancerous and cancerous lesions from 39 patients were imaged in vivo using a novel multispectral FLIM endoscope and processed to generate widefield maps of biochemical and metabolic autofluorescence biomarkers of oral precancer and cancer. Results: Statistical analyses applied to the quantified multispectral FLIM endoscopy based autofluorescence biomarkers indicated their potential to provide contrast between precancerous/cancerous vs. healthy oral epithelial tissue. Conclusion: To the best of our knowledge, this study represents the first demonstration of label-free biochemical and metabolic clinical imaging of precancerous and cancerous oral lesions by means of widefield multispectral autofluorescence lifetime endoscopy. Future studies will focus on demonstrating the capabilities of endogenous multispectral FLIM endoscopy as an image-guided surgical tool for positive margin detection during head and neck cancer resection surgery.