This study aimed to investigate the quality characteristics and bioactive properties of grape seed oil obtained from five different grape varieties (öküzgözü-Elaziğ, Syrah-çanakkale, Cabernet Franc-çanakkale, Pinot-Noir-Tekirdağ, and Merlot-Manisa) by cold press system. In addition, the antibacterial activity of grape seed oils was determined. The grape varieties significantly affected physicochemical properties, fatty acid and sterol composition of grape seed oils (P < 0.05). The protein and oil, total flavonoid, phenolic, and anthocyanin contents of grape seeds changed between 7.44-13.66 and 6.93-8.80%, were 254-1,436.67, 4,397.93-5,804.29, and 0.31-7.89 mg/g, respectively. The acidity, peroxide number and tocopherol amount of grape seed oils obtained by cold press method were determined as 0.67-2.74%, 10.45-22.03 meqO2/kg, and 2.04-11.595 mg /100 g oil, respectively. The most abundant fatty acids were palmitic, stearic, oleic and linoleic acids, and these values were 9.98-14.98, 3.61-6.97, 15.31-28.94 and 48.78-69.13% respectively. β-sitosterol exhibited major sterol for all varieties and ranged, 61.6 and 69.54%. All grape variety oils gradually showed antimicrobial activity at different ratios against Staphylococcus aureus (ATCC 2592), Escherichia coli (ATCC 25922), Salmonella enterica subsp. enterica serovar Enteritidis (ATCC 13076) and Aspergillus parasiticus DSM 5771. This study suggested that grape seed oil of selected varieties could be utilised as a good source of edible oil due to its antioxidant, essential fatty acids, sterol contents, and antibacterial properties.