Extensive use of pyrethroids has led to the development of resistance in several tick species, including Rhipicephalus annulatus. This study aimed to investigate the contribution of key detoxification enzymes to permethrin resistance and to evaluate whether chemical inhibition of these pathways could enhance acaricidal efficacy. Larval immersion tests were conducted on susceptible and resistant strains of R. annulatus to determine lethal concentrations of permethrin and to establish non-lethal concentrations of enzyme inhibitors targeting cytochrome P450 monooxygenases (piperonyl butoxide, PBO), glutathione S-transferases (diethyl maleate, DEM), and esterases (S,S,S-tributyl phosphorotrithioate, DEF). Combination bioassays were performed to assess mortality over time following co-exposure to permethrin and each inhibitor. The resistant strain exhibited substantially higher LC₅₀ and LC₉₀ values (472.66 and 3037.46 µM) compared to the susceptible strain (123.56 and 257.25 µM). Co-application of inhibitors enhanced permethrin-induced mortality in both strains, with the strongest effects observed for PBO and DEM. In contrast, DEF produced only modest increases in mortality. Regression analyses indicated that combinations with PBO and DEM were significantly more effective than DEF in increasing tick mortality. However, even with inhibitor treatment, mortality in the resistant strain did not reach the levels observed in the susceptible strain. These findings demonstrate that permethrin resistance in R. annulatus is mediated by a complex and multifactorial detoxification network. While inhibition of cytochrome P450 and glutathione S-transferases significantly enhances permethrin efficacy, it does not fully restore susceptibility, highlighting the involvement of additional metabolic and non-metabolic mechanisms and the need for integrated strategies targeting multiple resistance pathways.