The aerobic endospore-forming bacteria (AEFB) comprise species of Bacillus and related genera, allocated in the phylum Firmicutes. Although Bacillus spp. are among the first bacteria to be characterised, the wide diversity renders appropriate categorisation and generalisations challenging tasks. To determine genetic diversity, analyses at the molecular level are the most accurate. However, gene expression, morphological, biochemical, and physiological aspects must also be considered. The metabolism of bacteria is adapted to their natural environment or host. Thus, metabolic outlines can be used for identifying AEFB, form the basis of the formal description of bacterial taxa, and are strongly recommended for taxonomic purposes. This work addressed the biochemical and physiological profiles of 312 environmental AEFB—designated as SDF (Solo do Distrito Federal)—by performing 30 tests. Out of it, 246 were classified by 16S rRNA gene sequences. We summarised the phenotypic test relationships among selected SDF strains using a Pearson correlation-based clustering represented in heatmaps. In practice, biochemical and physiological profiles are often less discriminatory than molecular data and may be unstable because of the loss of traits. Though these test reactions are not universally positive or negative within species, they may define biotypes and be efficient strain markers, enhancing the accuracy of unknown sample identification. It can be also helpful in selecting the best represent the phenotypes of samples. Along with the other phenotypic and genotypic data, the present results will be of great importance for the robust classification of the SDF strains within the scope of the polyphasic approach.