The present investigation intended to evaluate the bacteriostatic and bactericidal abilities of clove, oregano and thyme essential oils against oral bacteria in planktonic and biofilm states. Furthermore, aiming to mimic everyday conditions, a toothbrush in vitro model was developed. Determination of the minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration and minimum biofilm eradication concentration were achieved using the microdilution procedure. To simulate the toothbrush environment, nylon fibers were inoculated with oral bacteria, which, after incubation to allow biofilm development, were submitted to contact with the essential oils under study. Thyme and oregano essential oils revealed promising antimicrobial effects, both in growth inhibition and the destruction of cells in planktonic and biofilm states, while clove essential oil showed a weaker potential. Regarding the toothbrush in vitro model, observation of the nylon fibers under a magnifying glass proved the essential oil anti-biofilm properties. Considering the effects observed using the in vitro toothbrush model, a realistic approximation to oral biofilm establishment in an everyday use object, a putative application of essential oils as toothbrush sanitizers to help prevent the establishment of bacterial biofilm can be verified.