Otitis media (OM) is a highly prevalent disease that can be caused by either a bacterial or viral infection. Because antibiotics are only effective against bacterial infections, blind use of antibiotics without definitive knowledge of the infectious agent, though commonly practiced, can lead to the problems of potential harmful side effects, wasteful misuse of medical resources, and the development of antimicrobial resistance. In this work, we investigate the feasibility of using a combined Raman scattering spectroscopy and low coherence interferometry (LCI) device to differentiate OM infections caused by viruses and bacteria and improve our diagnostic ability of OM. Raman spectroscopy, an established tool for molecular analysis of biological tissue, has been shown capable of identifying different bacterial species, although mostly based on fixed or dried sample cultures. LCI has been demonstrated recently as a promising tool for determining tympanic membrane (TM) thickness and the presence and thickness of middle-ear biofilm located behind the TM. We have developed a fiber-based ear insert that incorporates spatially-aligned Raman and LCI probes for point-of-care diagnosis of OM. As shown in human studies, the Raman probe provides molecular signatures of bacterial- and viral-infected OM and normal middle-ear cavities, and LCI helps to identify depth-resolved structural information as well as guide and monitor positioning of the Raman spectroscopy beam for relatively longer signal acquisition time. Differentiation of OM infections is determined by correlating in vivo Raman data collected from human subjects with the Raman features of different bacterial and viral species obtained from cultured samples.