A collection of bacteria belonging to the genus Bacillus were analysed by diffuse reflectance absorbance and attenuated total reflectance Fourier transform infrared (FT-IR) spectroscopy in the mid-infrared. The diffuse reflectance absorbance method is a rapid whole organism fingerprinting method, which generates a biochemical profile of the bacteria, where samples are presented to the FT-IR spectrometer dried on a metal carrier. The attenuated total reflectance FT-IR used in conjunction with a diamond attenuated total reflectance (ATR) accessory produces a biochemical profile of the surface chemistry of bacteria directly without the need for drying, and has not previously been used in the discrimination of bacteria. Principal component, discriminant function and hierarchical cluster analysis were performed on the data to discriminate the bacteria. The differentiation of the bacteria to species level was observed in both analyses however, it was concluded that the ATR FT-IR illustrated better sub-species differentiation of the microorganisms. This may imply that the total biochemical profiling infers discrimination to species level whereas strain specific markers are present on the cell surface chemistry.