This study was targeted to characterize the chemical composition and antibacterial properties of Daphne oleoides subsp. oleoides essential oil. The essential oil was analyzed and quantified by gas chromatography and gas chromatography/mass spectrometry. Additionally, the broth dilution method was used to evaluate its antibacterial activity against Bacillus cereus (ATCC 11778), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Streptococcus faecalis (ATTC 29212), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 25933), Proteus vulgaris (ATCC 13315), Pseudomonas aeruginosa (ATCC 27853), and Salmonella typhi Ty2 (ATCC 19430). Seventy-nine compounds were identified, representing 95.2% of the total oil. Nootkatone (18.5%), nootkatin (12.1%), and daphnauranol C (11.7%) were determined as the main constituents in the oil. Oxygenated sesquiterpenes were dominating in the oil (43.0%), followed by fatty acid derivatives (13.7%) and carbonylic compounds (9.6%). The minimal inhibitory/bactericidal concentrations of essential oils of D. oleoides were in the range from 25–100 μg/mL, which can be considered as high activity in comparison with the reference antibiotic which was active in the rangefrom 3.12–100 μg/mL. The greatest minimal inhibitory concentration value was determined as 25 µg/mL against both two Bacillus strains and S. epidermidis, B. cereus, B. subtilis, and S. aureus were the most sensitive strains against essential oils when compared with the minimal inhibitory concentrations of control antibiotic. Consequently, Daphne oleoides subsp. oleoides can be exploited as a source of natural antibacterial agents and nootkatone for the pharmaceutical, food, and agricultural industries.