Background: Lysinibacillus fusiformis is a Gram-positive, rod-shaped bacterium that can cause tropical ulcers, severe sepsis, and respiratory illnesses in humans. Objective: In this study, we analyzed cosmetic samples for the presence of human pathogenic microorganisms. Methods: Five unopened jars of exfoliating cream were examined initially by microbiological methods. Afterward, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS and 16S ribosomal RNA (rRNA) sequencing techniques were applied to characterize the recovered isolates. Results: Of the eight recovered Gram-positive bacterial subs, the VITEK® MS could provide genus-level identification to five subs and species-level identification to two subs (L. fusiformis with a 99.9% confidence value); one sub was unidentified. Subsequently, the deoxyriboneucleic acid sequencing of the 16S rRNA gene was done on an ABI 3500XL Genetic Analyzer for the confirmation of species identification. An analysis of sequencing data revealed a complete absence of genetic variation among the eight subs sequenced at this locus and confirmed the eight bacterial subs to be L. fusiformis, as their respective 16S rRNA sequences were identical to the available sequence in public domain (GenBank accession No. KU179364). Conclusions: Our results suggest that the VITEK MS and the 16S rRNA sequencing can be used for the identification of human pathogenic bacteria of public health importance. Highlights: We characterized eight isolates of Lysinibacillus spp. from cosmetics by MALDI-TOF MS and 16S rRNA sequence analyses.