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In the search for a healthy indoor climate, chemical cleaning agents are replaced by bacillus strains to create an indoor ecosystem in which these harmless bacteria clean the surfaces by consuming the organic molecules in dust and dirt. To optimise these ecosystems, it is important to monitor the bacterial growth and activity, but the reference methods are time consuming and involve a serious time delay. Therefore, the potential of fluorescence spectroscopy for monitoring the activity of bacillus strains was investigated in this study. We confirmed that fluorescence signals could be detected for Bacillus vegetative cells at a load above 108 cells mL−1. As the fluorescence emission was mostly in the 330–400 nm range after excitation in the 280–290 nm range, we could attribute this signal to the tryptophan residues. The Bacillus fluorescence signals could be distinguished from those released by pure l -tryptophan, plate count agar medium, and Bacillus spores, which were classified with 100% accuracy by PLSDA. In comparison to other microbes, bacillus strains, Escherichia coli (MG1655) and Saccharomyces cerevisiae were found to produce an indistinguishable peak at 340 nm in response to excitation at 280 nm. However, using a hybrid multivariate classifier involving PCA combined with a One-Class SVM on the area-normalised fluorescence emission spectra in response to excitation at 280 nm allowed footprints of Bacillus cells to be identified with a classification accuracy of 78%. • Bacillus subtilis can be detected at above 108 cell mL-1. • Fluorescence of bacillus cells is mainly generated by tryptophan residues. • A hybrid multivariate classifier can discriminate Bacillus strains from other microbes. [ABSTRACT FROM AUTHOR]