Hop essential oil (EO) generates interest for its antioxidant and antimicrobial properties, in addition to the volatile compounds that are responsible for the hop aroma in beer. Thus, the objective of this study was to evaluate the chemical composition, EO yield, and antibacterial activity of hop essential oil from hops of the Chinook variety against lactic acid bacteria (Lactobacillus brevis and Lactobacillus casei) at different times of extraction. EO extraction was performed by hydrodistillation at different times. By analyzing the chemical composition by gas chromatography and mass spectrometry, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined. The major compounds of hop EO were α-humulene, β-myrcene, and β-caryophyllene, and the extraction yields were 0.67, 0.78, and 0.85% mass of EO per mass of hops pelletized hops (m/m), for extractions of 90, 180, and 300min, respectively. The EO obtained in 90min was efficient against L. casei at 2.5mg/mL (MIC) and 5.0mg/mL (MBC), and the 300min one against L. brevis at 2.5mg/mL (MIC) and 25mg/mL (MBC). The antibacterial activity was affected by the chemical makeup of the oil, revealing that the hop EO extracted in 300min was the most efficient among the other extraction times.