The ability of normal Raman and surface-enhanced Raman scattering (SERS) to identify and detect bacteria has shown great success in recent studies. The addition of silver nanoparticles to bacterial samples not only results in an enhanced Raman signal, but it also suppresses the native fluorescence associated with biological material. In this report, Raman chemical imaging (RCI) was used to analyze individual bacteria and complex mixtures of spores and vegetative cells. RCI uses every pixel or a binned pixel group (BPG) of the Raman camera as an independent Raman spectrograph, allowing collection of spatially resolved Raman spectra. The advantage of this technique resides primarily in the analysis of samples in complex backgrounds without the need for physically isolating or purifying the sample. Using a chemical imaging Raman microscope, we compare normal RCI to SERS-assisted chemical imaging of mixtures of bacteria. In both cases, we are able to differentiate single bacterium in the Raman microscope's field of view, with a 60-fold reduction in image acquisition time and a factor of 10 increase in the signal-to-noise ratio for SERS chemical imaging over normal RCI. Copyright © 2010 John Wiley & Sons, Ltd.