Bacillus spp. can be regarded as a rare component of the nasal mucosa microflora. The aim of this study was to identify Bacillus spp. from the nasal mucosa of healthy adults living in the suburban area near Lublin using the matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF MS) system. A total of 11 bacterial isolates from the nasal specimens were cultured. The following species were identified using the routine microbiological methods: Staphylococcus aureus (3 isolates), S. epidermidis (1 isolate), S. intermedius (1 isolate) and Staphylococcus spp. (1 isolate). Moreover, 2 strains of Escherichia coli were isolated. Besides, 3 isolates of Bacillus spp. were found. These isolates were characterized by means of MALDI-TOF MS, resulting in highly specific mass spectral fingerprints and these were identified as B. pumilus, B. safenis and B. licheniformis. It was observed that all studied Bacillus spp. isolates only had the masses in common at 3864 ± 2, 7727 ± 2, and 14301 ± 4. The spectra of B. safensis and B. pumilus showed peaks at m/z 4914 ± 3, 6621 ± 3 and 14291 ± 2, which were absent in the spectrum of B. licheniformis. For B. safenis and B. pumilus, other potential biomarkers could be found at m/z 12620 and 16668, respectively.