Acinetobacter baumannii is one of the most important gram-negative microorganisms which lead to opportunistic hospital-acquired infections. A great part of the infections it causes is produced by strain resistant to all the antibiotics used. In our study, the Essential Oil (EO) of the Ziziphora tenuior L. antimicrobial and antibiofilm effects on multidrug resistant (MDR) A. baumannii isolates were researched. In addition, antioxidant, cytotoxic activity and chemical composition of EO were investigated. As a result of the gas chromatography-mass spectrometry (GC-MS) analysis of the Z. tenuior EO pulegone was found as the major component by 74.37%. It was observed when the antimicrobial activity of the EO was examined that it had >30 mm disc diffusion values. Minimal Inhibition Concentration (MIC) values were found between 0.6-1.25 mu L mL(-1) and Minimal Bactericidal Concentration (MBC) values were found between 2.5-5 mu L mL(-1). Minimal biofilm inhibition concentration (MBIC) values of the EO were found as 0.3-1.25 mu L m L-1 and Minimal Biofilm Eradication Concentration (MBEC) value as 5-10 mu L mL(-1). It was seen that the MIC value damaged the biofilm formations constituted by the A. baumannii strains by 51-84%. At 25, 12.5 and 6.25% EO concentrations, no cytotoxic appeared for the fibroblast cells in terms of the cytotoxic activities (p>0.05). Findings that were obtained in our study seem promising for the development of phytotherapeutic agents that could be used in the treatment of the MDR A. baumannii infections.