Biological degreasing system is a new technology based on the degradation capabilities of microorganisms to remove oil, grease, or lubricants from metal parts. No data is available about the potential biological health hazards in such system. Thus, a health risk assessment linked to the bacterial populations present in this new degreasing technology is, therefore, necessary for workers. We performed both cultural and molecular approaches in several biological degreasing systems for various industrial contexts to investigate the composition and dynamics of bacterial populations. These biological degreasing systems did not work with the original bacterial populations. Indeed, they were colonized by a defined and restricted group of bacteria. This group replaced the indigenous bacterial populations known for degrading complex substrates. Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, and Pantoea agglomerans were important members of the microflora found in most of the biological degreasing systems. These bacteria might represent a potential health hazard for workers.