Development of an available drug delivery system is of great interest considering the therapeutic effects of formulations with violate essential oils, especially assuming the fact that using essential oils as antimicrobial agents is a rather expanded in antimicrobial ther- apy nowadays. In this paper, the chitosan microparticles with encapsulated thyme essen- tial oil were prepared in the emulsion by cross-linking method. The effect of thyme oil and glutaraldehyde initial concentrations on particle size, morphology, and particle size distri- bution was investigated. In addition, the influence of these parameters on the encapsul- ation of thyme oil in chitosan microparticles, concerning thyme oil loadings and encapsul- ation efficiency was also tested. The particles showed a spherical shape with an average diameter of 4.71±1.42 to 13.65±4.34 μm, depending on the concentration of the essential oil and glutaraldehyde that were used. The diameter of microparticles appeared to inc- rease with increasing the thyme essential oil concentration, and decreased with the inc- rease of glutaraldehyde concentration. It was shown that the concentration of glutaral- dehyde did not affect the degree of encapsulation, but the increase in the initial concen- tration of thyme oil increased the degree of encapsulation of this essential oil in chitosan microparticles. All particles containing thyme essential oil, as well as essential oil of thyme itself showed significant antimicrobial activity against Staphylococcus aureus ATCC 25923, Esherichia coli ATCC 25922, Candida albicans ATCC 24433 and Enterococcus faecalis ATCC 25929. This study showed a great potential of the use of thyme essential oil as an anti- microbial agent, especially when encapsulated in a drug delivery system with controlled release of the active antimicrobial component.