Recent investigations have focused on the improvement of rapid and accurate methods to develop spectroscopic markers of compounds constituting microorganisms that are considered biological threats. Quantum cascade lasers (QCL) systems have revolutionized many areas of research and development in defense and security applications, including his area of research. Infrared spectroscopy detection based on QCL was employed to acquire mid infrared (MIR) spectral signatures of Bacillus thuringiensis (Bt), Escherichia coli (Ec) and Staphylococcus epidermidis (Se), which were used as biological agent simulants of biothreats. The experiments were carried out in reflection mode on various substrates such as cardboard, glass, travel baggage, wood and stainless steel. Chemometrics statistical routines such as principal component analysis (PCA) regression and partial least squares-discriminant analysis (PLS-DA) were applied to the recorded MIR spectra. The results show that the infrared vibrational techniques investigated are useful for classification/detection of the target microorganisms on the types of substrates studied.