Citrus aurantium amara (sour orange) belongs to one of the largest genera of the Rutaceae family. The species of this genus are consumed worldwide as fresh or in form of beverages. They include well-known crops lemons, oranges, mandarins, grapefruits, and limes. The industrial processing of these fruits produces high amounts of waste (around 50%) which is a valuable source of essential oils. Since they are produced mainly from peel, considered waste, these essential oils have great economic value. In that regard, the aim of this study was to evaluate the chemical composition of the essential oil obtained from the peel of Citrus aurantium amara, as well as to assess the biological effects by the means of antioxidant and antibacterial activity. Results of GC and GC/MS analysis characterized this EO as a valuable source of limonene found in the amount of 90.4% of the total. Results of antioxidant activity indicate better inhibition of ABTS•+ (44.93 ±1.45%) compared to the DPPH• (11.03 ±1.08%). Moreover, the results of the antimicrobial assessment using the disc diffusion method displayed low inhibition potency of this essential oil towards G+ and G− bacteria and yeast strains.