Aim: The present study focused on the evaluation of acaricidal efficacy of ethanolic extract of fruits of Datura stramonium against cattle tick Rhipicephalus microplus in-vitro. The ethanolic extract of fruits of Datura stramonium were analysed for the presence of phytochemicals responsible for antitick activities. Methodology: The fruits of Datura stramonium were dried, blended, powdered and extracted in ethanol using Soxhlet’s apparatus. In-vitro tests like Adult Immersion Test and Larval Packet Test were utilized to assess the effect of plant extract by utilizing engorged R. microplus female. Reproductive Index, Inhibition of oviposition, adult and larval mortality of female ticks were observed. The phytochemicals present in the ethanolic fruits extract of D. stramonium were assessed by Gas chromatography-Mass spectroscopy and Fourier Transform Infra-red Spectroscopy. Results: The present study indicated anti tick activities of ethanolic extract of Datura stramonium fruit against Rhipicephalus microplus by reporting a mortality of 66.67%, Reproductive Index (0.11-0.16), Inhibition of oviposition ( 70.62%) and larval mortality (65%) at 100 mg ml-1 and LC50 value was determined to be 61.66 mg ml-1. There was dose dependant mortality of adult and larva and inhibition of reproduction. The ethanolic fraction's spectral examination revealed 31 components, with the presence of 2(4H)-Benzofuranone, 5,6,7,7a-(7.24), Hexadecanoic acid, methyl ester (9.16), 9,12,15-Octadecatrienoic acid, methyl ester (9.35), 9,12,15-Octadecatrienoic acid (15.17), 2-Propenoic acid, 3-phenyl-, methyl ester (32.87) as major phytocompounds. Interpretation: These results showed that ethanolic extract of Datura stramonium fruits are source of phytochemicals with antitick activities. The findings would be useful to the pharmaceutical industries for developing of ecologically friendly natural acaricides. Key words: Acaricides, Datura stramonium, Ethanolic extract, Rhipicephalus microplus, Ticks