Extraction of essential oil is one of the oldest methods of recycling plant biomass, using pine needles as a valuable raw material and thus reducing the amount of biowaste. The composition of essential oil mainly depends on the type of plant material. In the present study, Black Pine, Norway Spruce, Blue Spruce and Pančić Spruce (Serbian spruce) were used. The chemical composition and microbiological activity of essential oils extracted by the method of hydrodistillation by Clevenger and Soxhlet extraction were researched. The chemical composition was analyzed by thin layer chromatography, gas chromatography with mass spectrometry and infrared spectroscopy with Fourier transform. The main components of Black Pine (Pinus nigra) essential oil are α-pinene (34.16%) and germacaren D (29.79%). The analysis of essential oils extracted by hydrodistillation of Spruce needles (Picea pungens, Picea abies and Picea omorika) showed great similarity in chemical composition between those three species. Dominant component in all three samples was bornyl acetate in concentrations from 21.97 to 53.19 percent. The antimicrobial activity was tested against Gram-negative and Gram-positive bacterial cultures and yeast of the genus Candida. The intensity of the inhibitory effect of the essential oils used varied depending on the microbial culture used.