We report on the spectroscopic investigation of common bacteria encountered in biopharmaceutical industries with spectroscopic definition and specificity using mid-infrared laser spectroscopy. This study describes the detection of three different bacteria species using quantum cascade laser spectroscopy coupled to a grazing angle probe (QCL-GAP). Stainless steel substrates were used as support for the bacterial samples. QCL-GAP spectroscopy was assisted by multivariate analysis (MVA) to assemble a powerful spectroscopic technique with classification, identification, and quantification resources. The bacterial species analyzed, Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, were used to challenge the technique’s capability to discriminate microorganisms from the same family. Principal component analysis and partial least squares-discriminant analysis differentiated between the bacterial species using QCL-GAP. Spectral differences in the bacterial membrane were used to determine if these microorganisms were present in the samples analyzed. Results herein provided effective discrimination for the bacteria under study with high sensitivity and specificity values.