Pyrethroid insecticides have widely been used to control the house fly, Musca domestica. Toxicological and biochemical studies were conducted for monitoring housefly tolerance to three pyrethroid insecticides λ-cyhalothrin, deltamethrin and α-cypermethrin. Based on the LC₅₀ values of third larval instar laboratory (LS) and field strains (FS) exposed for 72 h using a poisonous media technique, flies showed resistance ratios ranging from 4.06 to 7.59-fold. The highest house fly population homogeneity was observed with deltamethrin in LS (2.13) and α-cypermethrin in FS (1.66). The biochemical evaluation was conducted in the third larval instar after exposure to estimated LC₅₀ values of the tested insecticides on both strains. The protein content of FS was significantly higher in the control and λ-cyhalothrin and decreased following treatment with deltamethrin and α-cypermethrin. In contrast, mixed function oxidase enzymes (MFOs) were significantly higher in FS under all treatments, while β-esterase was significantly highest in λ-cyhalothrin FS. The α-esterase activity declined significantly with different pyrethroid treatments of FS. Glutathione-S-transferase enzyme (GST) activity was highest in all treatments of FS, except α-cypermethrin was lower in LS. The activity of Acetylcholinesterase enzyme (AChE) following pyrethroid treatments decreased significantly in FS compared with LS and control treatments. Carboxylesterase was significantly higher in all pyrethroid treatments of FS. Significant interactions were observed between strains and pyrethroid treatments. The development of pyrethroid resistance in FS, and the role of mixed-function oxidases and β-esterase in the degradation of different pyrethroids, in addition to, β-esterase and GST with λ-cyhalothrin detoxification, may explain the highest tolerance ratio (7.59).