The strongest inhibition activity against Gram-negative Yersinia enterocolitica, and Gram-positive Clostridium perfringens, Enterococcus faecalis, and Staphylococcus aureus subs. The Gram-negative Escherichia coli was strongly inhibited by essential oil fractions from 2-5 min and 10-30 min, followed by oil fraction from 0-2 min. Myrcene and linalool were the highest in 0-2 min fraction, β-caryophyllene, β-copaene, β-farnesene, and α-humulene were highest in fractions from middle of distillation, whereas α- bergamotene, γ-muurolene, β- and α-selinene, γ- and δ-cadinene, caryophyllene oxide, humulne epoxide II, τ-cadinol, and 6-pentadecen-2-one were highest in 120-180 or 180-240 min fractions. Monoterpenes were eluted early, while sequiterpenes were eluted late. Essential oil (EO) fractions had different chemical profile. The oil eluted during the first hour was 83.2%, 9.6% during the second hour, and only 7.2% during the second half of the distillation. Fractions yielded 0.035 to 0.313% essential oil, while control yielded 1.47%. Nonlinear regression models described the DT and essential oil compounds relationship. The control was a 4-h non-interrupted distillation. We hypothesized that compound mixtures eluted sequentially and captured at different timeframes during the steam distillation process of whole hop cones would have differential chemical and bioactivity profiles. The profile and bioactivity of hops ( Humulus lupulus L.) essential oil, a complex natural product extracted from cones via steam distillation, depends on genetic and environmental factors, and may also depend on extraction process.