OBJECTIVE We aim to assess the antibacterial effectiveness of probiotic cleaning in a dental clinic at the University Dental Hospital Sharjah (UDHS), UAE. METHODS The current cleaning protocol of UDHS was evaluated by the surface swabbing of three dental clinics routinely cleaned using regular chemical disinfectants. Then, a new probiotic cleaning solution containing Bacillus subtilis was applied for 3 weeks in a selected clinic. Bacteria were grown onto selective culture media for colony counting from surfaces cleaned with probiotic solution compared to those obtained from the same surfaces cleaned with the regular chemical solutions. Isolates identity was confirmed by biochemical tests or polymerase chain reaction. RESULTS There was a significant reduction of the bacterial counts of various bacterial species (Staphylococci, Streptococci, and Gram-negative rods) from almost all the surfaces in the dental clinic after the application of the probiotic solution compared to the same surfaces cleaned with the regular chemical solutions. However, the antibiotic resistance rates were not significantly reduced within the short period of 3 weeks of using the new probiotic cleaning product. CONCLUSIONS This study demonstrated that the use of probiotic cleaning is effective in reducing microbial growth in dental settings. This approach may be tested further to examine the long-term effect and to evaluate the opportunity of applying this novel biotechnology as part of the infection control routine in dental settings instead of the chemical disinfectants which are known to cause serious health problems. This is the first study testing the application of probiotic-based solution in dental settings.