The present study was performed with an objective to determine acaricidal activity of Citrus limetta seed oil (CLO) against cattle tick Rhipicephalus microplus. The mode of action of CLO was determined by evaluating biochemical enzymatic levels in treated ticks. CLO was obtained by extracting with n-hexane as solvent in soxhlet apparatus. The active compounds in CLO were analyzed through Gas chromatography. Acaricidal activity of CLO was evaluated using larval packet test (LPT) and adult immersion test (AIT). Results of chromatographic analysis showed that palmitic acid, stearic acid, α-Tocopherol and bergamol were present as major active constituents along with other compounds. The lethal concentration (LC₅₀) of CLO was found to be 2.87% and 3.96% for larvae and adult of R. microplus ticks, respectively. In vitro acaricidal bioassay revealed 100% mortality of ticks in both LPT and AIT at 12.5% dose. CLO significantly (p < 0.001) reduced oviposition rate, hatching ability of eggs and reproduction efficiency of ticks. Biochemical analysis of ticks homogenate showed different enzymatic levels. The results revealed that CLO disturbed protective enzymes functions by reducing level of SOD, GST, MAO and AChE but increasing NOS level in ticks as compared with control group. Thus, CLO may be used as an effective acaricidal agent in controlling R. microplus ticks.