Abstract Origanum majorana L. (Lamiaceae) is a plant that is used in gastronomy and natural medicine. The plant material used in this study was collected at San Isidro de Apartaderos, Mérida State. A yield of 0.6% of essential oil was obtained by hydro-distillation using a Clevenger trap. The main constituents found were: cis-sabinene hydrate (30.2%), terpinen-4-ol (28.8%), γ-terpinene (7.2%), α-terpineol (6.9%), trans-sabinene hydrate (4.4%), linalyl acetate (3.8%), and α-terpinene (3.6%). The essential oil was also fractionated over a silica gel dry column. Two main fractions were isolated, the first containing 67.6% of cis-sabinene hydrate and the second 72.8% of terpinen-4-ol. The antibacterial activity of the oil was determined using the agar diffusion method and it was found that it was active against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Klebsiella pneumoniae. Antibacterial activity of the fractions obtained by dry column chromatography was also tested. It was found that the fraction rich in cis-sabinene hydrate was more active than the one rich in terpinen-4-ol. It was concluded that in the essential oil of Origanum majorana, cis-sabinene hydrate is the more important compound responsible for inhibition of bacterial growth.