The bacteria fatty acid profile has been extensively studied for taxonomic classification purposes, since bacteria, in general, contain particular and rare fatty acids, compared with animal and plant tissues. As for any real-world sample type, the development of rapid and reliable methods for (i) sample identification (in this case, bacterium type), and (ii) constituent identification (in this instance, the fatty acid profile) is desirable. In this research, a half-an-hour procedure, to analyze bacteria, was developed: a 2-min one-step sample preparation step was followed by a relatively fast comprehensive 2D GC-MS separation (25 min). Furthermore, dedicated MS libraries were constructed for the identification of bacteria and fatty acids. Finally, data processing, only qualitative at this stage, was carried out with the support of a novel comprehensive 2D GC software.