Near infrared spectroscopy is a widespread technique in analytical chemistry. How- ever, recently, there has been a growing interest in the usage of the NIR spectroscopy in microbio- logical analysis. Due to the improvement of the experiment setup and an increase in the efficiency of the evaluation methods, the knowledge of the absorption coefficient of a sample can be very beneficial. The purpose of this study is to introduce problems that relate to the measurement and handling of absorption coefficients. The molecular bonds presented in the examined sample cause absorption of the incident light. When the energy quantum from a source of light equals the energy necessary for the transition of a bond to a higher vibration level, the light is absorbed. Considering this effect, it is possible to identify the chemical composition of a sample. Recent studies [1] have proved that the chem- ical composition of different bacteria species is sufficiently diverse, and thus the bacteria can be identified using a NIR spectrum. The absorption coefficient is mostly used in the Lambert-Beer- Bouguer law. This equation expresses how the optical intensity of a light wave is exponentially reduced along the beam’s path through the sample. In the case of NIR spectroscopy, the out- put light intensity is measured. The multiplicative scatter and other unwanted influences of the incoming radiation make direct measurement difficult. For correct computing of the absorption coefficient, it is necessary to separate the physical light-scattering effect from the chemical light absorbance constituted by vibrations of a molecule’s bonds. The absorption coefficient is a func- tion of the energy of photons (wavelength), and its value depends on the position and amplitude of the fundamental, overtone, and combination types of molecular bond absorption. Knowledge of the absorption coefficient is important due to optimizing the measurement tech- nique and statistics methods. Because any straightforward measuring of bacteria cells requires a special approach, prior knowledge of the absorption coefficient can help to reduce costs in terms of money and time.