The use of synthetic pesticides has led to many problems in recent years. Synthetic pesticides have caused contamination of the environment with toxic residues in many regions around the world, as well as side-effects on non-target insects and other organisms, and an increase in the numbers of resistant pest species to pesticides. So it needs the use of safe, cheap, and more eco- friendly pesticides for mite control. Pesticides derived from plants have less harmful effects on mammals and are decomposed rapidly in the environment. The use of plant compounds can be considered as reliable alternatives in pest management. Tetranychus cinnabarinus Boisd (Tetranychidae) is one of the most common pests in greenhouses, causing damage with creating yellow and brown spots, destroying chlorophyll, spinning wrap, disrupting photosynthesis. In this study, the effect of contact toxicity of Eruca sativa (Miller) (Brassicaceae) oil and its methanol extract were investigated on adult female T. cinnabarinus at five concentrations and three replications set at 25 ± 2 °C and 65 ± 5% RH and 16: 8 h (light: dark). The mortality was evaluated 24 h after treatment. Probit analysis was used to determine LC50 values. Results showed that the LC50s of E. sativa oil and the methanol extract were 3080 and 5674 ppm, respectively. Measurement of isothiocyanate content in the oil and methanolic seed extract by high performance liquid chromatography (HPLC) showed that the amount of isothiocyanates in oil was higher than its methanol extract. Also, the content of fatty acids in the oil and the methanol extract using the gas chromatography showed that the amounts of palmitic, stearic, oleic and linoleic acids in the oil were higher than those in methanolic extract. Due to the low amount of isothiocyanate and fatty acids in the methanol extract, the high acaricidal activity of E. sativa oil can be attributed to the type and content of isothiocyanate and fatty acids. Therefore, our findings show that the extraction technology of E. sativa oil plays a decisive role in its efficacy.