Chronic persistent middle ear infections (otitis media) can lead to cholesteatoma, an ingrowth of multi-layered keratinizing squamous epithelium, which requires complete removal in a surgical procedure. However, intraoperative detection is difficult and may result in remaining inflammatory tissue, leading to recurrence. Fluorescence lifetime imaging microscopy (FLIM) is a technique to measure and image the fluorescence lifetime (FLT) of fluorescent molecules. To date, there is no commercial system available to monitor FLT of intrinsic fluorophores during middle ear surgery. In this work, we performed FLIM on cryopreserved human middle ear tissue samples: two non-inflammatory biopsy specimens (external auditory canal and middle ear mucosa), otitis media and cholesteatoma. The thin-sliced tissue samples were examined with FLIM using excitation wavelengths of 375 nm and 473 nm. Results showed different FLTs among the various tissue types. Differences in the FLTs were observed in the 500-575 nm emission range with the 473 nm excitation. This could either be related to the presence and amount of enzymes in the cells or also refer to the structural diversity in the human middle ear tissues and the respective content of, for instance, collagen, elastin and keratin. Additional series of experiments are needed for more detailed analyses on the possible sources of the emission signals. These initial measurements should provide an overview of the occurring endogenous autofluorescence and the FLTs of different human middle ear tissue, in order to distinguish tissue types.