Bacillus are aerobic spore-forming bacteria that are known to lead to specific diseases, such as anthrax and food poisoning. This study focuses on the characterization of these bacteria by the detection of lipids extracted from 33 well-characterized strains from the Bacillus and Brevibacillus genera, with the aim to discriminate between the different species. For the purpose of analysing the lipids extracted from these bacterial samples, two rapid physicochemical techniques were used: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography in conjunction with mass spectrometry (LC-MS). The findings of this investigation confirmed that MALDI-TOF-MS can be used to identify different bacterial lipids and in combination with appropriate chemometrics allows for the discrimination between these different bacterial species, which was supported by LC-MS. The average correct classification rate (CCR) for the 7 species of bacteria was 62.23% and 77.03% based on MALDI-TOF-MS and LC-MS data, respectively. The Procrustes distance for the two datasets was 0.0699 indicating that the results from the two techniques were very similar. In addition, we also compared these bacterial lipid MALDI-TOF-MS profiles to protein profiles also collected by MALDI-TOF-MS on the same bacteria (Procrustes distance 0.1006). The level of discrimination between lipids and proteins was equivalent and this further indicates the potential of MALDI-TOF-MS analysis as a rapid, robust and reliable method for the classification of bacteria based on different bacterial chemical components.