ABSTRACT Rosemary (Rosmarinus officinalis L) essential oil (REO) has an inhibitory effect against pests and is regarded as a safe alternative to synthetic acaricides. This study investigated the acaricidal and repellent activity of REO and underlying mechanisms against Aleuroglyphus ovatus, an important mite pest of stored products. Gas Chromatography-Mass Spectrometry analysis of REO indicated that the compounds of 1,8-cineole (25.20%) was the highest. REO at 13% concentration provided the highest repellent effect (92.67%) on A. ovatus, and the lowest repellent activity (48.67%) was at 0.8% concentration. Microscopic observations revealed that the mite epidermis started to shrink after treatment with REO at 6 h post-treatment. The enzyme level of catalase (CAT), acetylcholinesterase (AChE) and superoxide dismutase (SOD) decreased significantly (P < 0.05), whereas there was a significant increase (P < 0.05) in nitric oxide synthase (NOS) and glutathione-s-transferases (GST) activities in A. ovatus treated with REO. In the molecular docking study, camphor and 1,8-cineole, the main monoterpenes of REO, bind more closely with CAT and AChE, and PI-conjugated interaction, hydrogen bonding, and hydrophobic were found by molecular docking 3D model. Overall, our results indicate the potential of REO as an effective botanical acaricide to manage A. ovatus.