Thymbra spicata has been used traditionally as herbal tea for the treatment of some diseases such as asthma, bronchitis, respiratory, coughs, and sore throat infection in Anatolia. In the current study, the essential oil of T. spicata (EOTS) were investigated for their antimicrobial, anti-biofilm activity and chemical composition. The chemical composition of Thymbra spicata was analyzed by gas chromatography-mass spectrometry (GC-MS). Antimicrobial and anti-biofilm properties were determined using broth microdilution methods. According to the results of the present study, the GC-MS profile highlights that the mains compounds were found grandisol 21.99%, cadinol 6.45%, lavandulol acetate 4.39%. The antibacterial activity of the EOTS was evaluated by the micro-dilution method against multidrug resistant Pseudomonas aeruginosa. EOTS produced inhibitory effects against the tested strains. It inhibited 11 clinical isolates of against P. aeruginosa with Minimum Inhibitory Concentration (MIC) values in the range of 1.56-12.5 μl/mL. Minimum Biofilm Inhibitory Concentration (MBIC) value was found to be range of 3.12-25 (μl/mL) while the Minimum Biofilm Eradication Concentration (MBEC) value was found to be 6.25-25 (μl/mL). MIC value of the EOTS was applied onto the bacterial biofilm formations. It was seen that biofilm metabolism was reduced by 2.0-51.6%. The findings of this study show that EOTS has antibacterial and anti-biofilm activity against P. aeruginosa isolates. Thus, essential oil of T. spicata may be useful a potential source for the treatment of multidrug resistant P. aeruginosa infections and biofilms.