Pseudomonas aeruginosa is an opportunistic pathogen that causes acute or chronic infections. Acute infections are caused by the planktonic form of the bacterium. Chronic infections most commonly develop in patients with chronic wounds and immunocompromised patients, and are caused by biofilm-developing cultures. Numerous mechanisms of resistance, including biofilm, developed by this bacterium contribute to its antibiotics resistance, which further complicates the choice of effective therapy. Today, the challenge is to find new possibilities for the treatment of infections caused by P. aeruginosa, but also to achieve the best possible effect of classical therapy. Essential oils are substances that are known to possess antimicrobial effect, but the mechanisms of their action have not yet been thoroughly investigated. Recent research shows that certain essential oils also have an antibiofilm effect which may contribute to advances in therapy for the treatment of chronic P. aeruginosa infections. The aim of this study was to examine the effect of Micromeria thymifolia (Scop.) Fritsch essential oil and pulegone on planktonic cell growth and biofilm formation of P. aeruginosa ATCC 27853, alone and combined with tobramycin after biofilm formation. The effect on planktonic cell growth was determined by measuring the optical density of the culture at 570 nm. The amount of biofilm formed was determined by measuring the optical density of the culture at 540 nm, following the crystal violet staining. The independent effect of the M. thymifolia (Scop.) Fritsch essential oil and pulegone on the biofilm was additionally shown as a biofilm index. The results showed that the M. thymifolia (Scop.) Fritsch essential oil and pulegone can in narrow range of subinhibitory concentrations effectively inhibit the growth of P. aeruginosa ATCC 27853 planktonic cells, alone and combined with tobramycin. It has also been shown that the M. thymifolia (Scop.) Fritsch essential oil and pulegone can in narrow range of subinhibitory concentrations inhibit biofilm development. In combination with tobramycin, the M. thymifolia (Scop.) Fritsch essential oil at most concentrations antagonized the effect of tobramycin on the further development of the formed biofilm, and pulegone had an antagonistic effect at all observed concentrations. This paper will provide new information and contribute to further research into new treatment options for infections caused by P. aeruginosa, but also to the preservation of existing therapy.