The formation of antibiotic-resistant strain biofilms in tympanostomy tubes results in persistent and refractory otorrhea. In the present study, we investigated the in vitro antibiofilm activity of thymol against biofilms formed by methicillin-resistant (MRSA) and ciprofloxacin-resistant (CRPA), using live and dead bacterial staining and adhesion, biofilm formation, biofilm eradication, and biofilm hydrolytic activity assays. The antibiofilm activity of thymol against tympanostomy tube biofilms formed by MRSA and CRPA strains was examined using a scanning electron microscope. In response to thymol treatment, we detected significant concentration-dependent reductions in the viability and adhesion of MRSA and CRPA. Exposure to thymol also inhibited the formation of both MRSA and CRPA biofilms. Furthermore, thymol was observed to enhance the eradication of preformed mature biofilms produced by MRSA and CRPA and also promoted a reduction in the rates of MRSA and CRPA hydrolysis. Exposure to thymol eradicated extracellular polysaccharide present in the biofilm matrix produced by MRSA and CRPA. Additionally, thymol was observed to significantly eradicate MRSA and CRPA biofilms that had formed on the surface on tympanostomy tubes. Collectively, our findings indicate that thymol is an effective inhibitor of MRSA and CRPA biofilms, and accordingly has potential utility as a therapeutic agent for the treatment of biofilm-associated refractory post-tympanostomy tube otorrhea resulting from MRSA and CRPA infection.