Multispectral imaging has recently shown good performance in determining information about physiology, morphology, and composition of tissue. In the endoscopy field, many researches have shown the ability to apply multispectral or narrow-band images in surveying vascular structure based on the interaction of light wavelength with tissue composition. However, there has been no mention to assess the contrast between other components in the middle ear such as the tympanic membrane, malleus, and the surrounding area. Using CT, OCT, or ODT can clearly describe the tympanic membrane structure; nevertheless, these approaches are expensive, more complex, and time-consuming and are not suitable for most common middle ear diagnoses. Here, we show the potential of using the multispectral imaging technique to enhance the contrast of the tympanic membrane compared to the surrounding tissue. The optical absorption and scattering of biological tissues constituents are not the same at different wavelengths. In this pilot study, multiwavelength images of the tympanic membrane were captured by using the otoscope with LED light source at three distinct spectral regions: 450 nm, 530 nm, and 630 nm. Subsequently, analyses of the intensity images as well as the histogram of these images point out that the 630 nm illumination image features an evident contrast in the intensity of the tympanic membrane and malleus compared to the surrounding area. Analysis of such images could facilitate the boundary determination and segmentation of the tympanic membrane (TM) with high precision.