The aim of the present study was the chemical characterization of some traditionally used and therapeutically relevant essential oils (thyme, eucalyptus, cinnamon bark, clove, and tea tree) and the optimized microbiological investigation of the effect of these oils on clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). The chemical composition of the oils was analyzed by TLC, and controlled by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The antibacterial effect was investigated using a TLC-bioautographic method. Antibacterial activity of thyme, clove and cinnamon oils, as well as their main components (thymol, carvacrol, eugenol, and cinnamic aldehyde) was observed against all the bacterial strains used in this study. The essential oils of eucalyptus and tea tree showed weak activity in the bioautographic system. On the whole, the antibacterial activity of the essential oils could be related to their most abundant components, but the effect of the minor components should also be taken into consideration. Direct bioautography is more cost-effective and better in comparison with traditional microbiological laboratory methods (e.g. disc-diffusion, agar-plate technique).