The main aim of this study was to evaluate the oils from chamomile seeds as a new source of bioactive compounds suitable for human consumption. A green extraction technique with supercritical carbon dioxide (sc-CO2) at pressures up to 450 bar and temperatures up to 60 °C was employed for the production of a high amount of biologically active oil. Additionally, exhausted waste material was re-extracted using sc-CO2 with the addition of ethanol. By optimization in operating pressure, temperature, production cost, fraction of milled seeds, and co-solvent addition, the amount of separated chamomile oil increased from 2.4 to 18.6% and the content of unsaturated fatty acids up to 88.7%. Oils contained α-bisabolol oxide A and B in amounts up to 1.4%. Linoleic acid was detected in an amount up to 711.1 mg/g and α-linolenic acid up to 27.5 mg/g. The total phenolic content in separated oil reached 80.4 mg GAE/g while the total flavonoid content reached 11.6 mg QE/g. The obtained chamomile oils showed antioxidant activity with an IC50 of up to 3.9 mg/mL. Among the 23 tested microorganisms, the antimicrobial activity of oils was the most pronounced against Gram-positive bacteria. The cytotoxic activity of oils was tested on normal and cancer-derived cell lines. Results indicated a significant potential for oil from chamomile seeds, produced in an eco-friendly manner, as a functional food. Keywords: Antimicrobial activity; Antioxidant activity; Biological activity; Total phenolic content; Fatty acid composition; Linoleic acid