To address the need for a fast and sensitive method for the detection of bacterial contamination in solutions, the use of Fourier-transform near infrared (FT-NIR) spectroscopy and multivariate pattern recognition techniques was evaluated. The complex cellular composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that might be useful for identification and sub-typing. Bacteria including strains of Escherichia coli spp., Pseudomonas aeruginosa, Bacillus spp. and Listeria innocua were evaluated. The harvested cells were treated with ethanol (70% v/v) to reduce the safety concerns when evaluating pathogenic strains. The bacterial cells were concentrated on an aluminum oxide membrane to obtain a thin bacterial film. Spectra were collected by FT-NIR by using a diffuse reflection-integrating sphere. This simple membrane filtration procedure generated reproducible FT-NIR spectra that can be used for rapid discrimination among closely related strains. Principal Component Analysis (PCA) of transformed spectra in the 5000-4000 cm-1 region exhibited clusters that discriminated between bacteria species at levels < 1 mg wet cells weight (∼ 106-107 CFU/mg). Variations in the growth conditions of the bacteria substantially affected the FT-NIR spectra and diminished the ability of PCA to differentiate among strains; this underscores the importance of developing robust sampling protocols. FT-NIR in conjunction with multivariate techniques can be used for the rapid and accurate evaluation of potential bacterial contamination in liquids with minimal sample manipulation.