The spinose ear tick Otobius megnini is a causative agent of otoacariasis in horses and jockeys in the Nuwara Eliya racecourses. The present study aimed at investigating the status of acaricide resistance and underlying mechanisms in this tick species under laboratory conditions. Live ticks collected from the ear canal of horses were used to establish a laboratory colony. Larval bioassays were carried out with an organophosphate (malathion), pyrethroids (flumethrin and permethrin), and an organochlorine (DDT) using Larval Packet Tests (LPT). The LC 50 (24 h) values for malathion, flumethrin, permethrin and DDT were recorded as 0.11%, 0.0003%, 0.008% and 0.124%, respectively, and the respective values for LC 90 were 0.722%, 0.004%, 0.300% and 3.049%. Biochemical assays revealed that the mean specific activity levels of esterase, glutathione S-transferases (GSTs), and monooxygenase amounts were 0.064 ± 0.008 μmol min -1 mg -1 , 0.104 ± 0.050 μmol min -1 mg -1 , and 0.068 ± 0.020 equivalent units of cytochrome P450, respectively. The percentage remaining activity of the propoxur-inhibited Acetylcholinesterase (AChE) was 33.88 ± 13.60%. Our data indicated that the O. megnini population of Nuwara Eliya, Sri Lanka was most susceptible to flumethrin and showed resistance to DDT. Development of resistance to malathion through increased activity of esterase and insensitive AChEs was evident. Since the activities of monooxygenases and GSTs were not enhanced, permethrin and DDT resistance may have developed through target site insensitivity.