Autofluorescence (AF) imaging is a powerful tool for the detection of (pre-)neoplastic lesions in the bronchi. Several endoscopic imaging systems exploit the spectral and intensity contrast of AF between healthy and (pre-)neoplastic bronchial tissues, yet, the mechanisms underlying these contrasts are poorly understood. In this report, the effect of formalin fixation on the human bronchi AF, hence on the contrast, was studied by spectrofluorometric point measurements and DAFE (Diagnostic AutoFluorescence Endoscopy) broad field imaging. Generally, formalin-fixed samples have higher AF intensity than in vivo, whereas the emission spectral shape is similar. Additionally, the spectrofluorometric data showed a moderate decrease of the AF intensity on (pre-)neoplastic lesions relative to the healthy bronchial samples. However, this decrease was lower than that reported from in vivo measurements. Neither spectral measurements nor imaging revealed spectral contrast between healthy bronchial tissue and (pre-)neoplastic lesions in formalin. These results indicate that epithelial thickening and blood supply in the adjacent lamina propria are likely to play a key role in the generation of the AF contrast in bronchial tissues. Our results show that the AF contrast in bronchial tissues was significantly affected by standard, 10% buffered, formalin fixation. Therefore, these samples are not suited to AF contrast studies.