The formation of biofilms is determined by the type of microrganisms, availability of nutrients and the characteristics of substrate. In the present study, we aimed to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry profiling as a methodology to monitor Stenotrophomonas maltophilia biofilm development and evaluate the antibacterial, anti-biofilm and antioxidant activity of Mentha piperita and Pimpinella anisum essential oils against S. maltophilia. Biofilms were grown within polypropylene tubes containing a glass slide and wooden toothpick, and were cultivated 3, 5, 7, 9, 12 or 14 days of inoculation and with essential oils experiment also. Planktonic cells were obtained separately by centrifugation as control. MALDI-TOF experiment were performed, one by collecting biofilms from both the glass slide and the wooden toothpick external surface, and the other by acquiring biofilms from these surfaces after essential oils influence. The molecular results showed that MALDI profiling is able not only to distinguish between different biofilm stages, but it is also appropriate to indicate when the biofilm cells are released at the dispersion stage, which occurred first on glass slide and wooden toothpick. Finally, the present study pointed out that MALDI profiling may emerge as a promising tool for the biofilms formation and control.