A hyperspectral microscope imaging (HMI) method, which provides both spatial and spectral information of bacterial cells, was developed for foodborne pathogen detection. The AOTF-based hyperspectral microscope imaging system can be effective for characterizing spectral properties of biofilms formed by Salmonella enteritidis as well as Escherichia coli. The intensity of spectral images and the pattern of intensity distribution of spectra varied with system parameters (integration time as well as gain) of the HMI system. Preliminary results demonstrated that determination of optimum parameter values of the HMI system and the integration time, which must be no more than 250 ms, are important for quality image acquisition from biofilms formed by S. enteritidis. Among the 89 contiguous spectral images between 450 and 800 nm, the intensity of images at 458, 494, 522, 550, 574, 590, and 670 nm were distinctive for biofilm of S. enteritidis, whereas the intensity of spectral images at 546 nm was distinctive for E. coli with dark-field illumination with a metal halide light source. For more accurate comparison of intensity from spectral images, a calibration protocol of quantitative intensity comparison needs to be developed to standardize image acquisition using neutral-density filters and multiple exposures. For the identification or classification of unknown foodborne pathogen samples, ground truth region-of-interest pixels need to be selected for spectrally pure fingerprints from various foodborne pathogens such as E. coli and Salmonella species.