Culex pipiens is considered the potential vector of West Nile virus outbreaks that have affected Tunisia. The objective of the present study was to evaluate the fenitrothion insecticide resistance status of Culex pipiens pipiens in three populations of insects from three Tunisian states. Standardized method bioassays were conducted to shed light on fenitrothion susceptibility status in field Culex pipiens pipiens populations.The cross-resistance between fenitrothion and propoxur, and the polymorphism of over-produced esterases and AChE 1 were also investigated. Resistances to fenitrothion were moderate and low in sample # 2 and 3 (12.9 and 7.2, respectively). The highest resistance ratio was observed in populations collected in Northern Tunisia (sample # 1) with a rate of 60.9 fold at the LC₅₀ level. The use of a synergist suggested the non-involvement of CYP450, esterases and/or glutathione-S-transferase in resistance of Culex pipiens pipiens to the tested insecticide. However, the biochemical investigation of detoxification enzymes using starch electrophoresis showed the involvement of four esterases, including A2-B2, A4-B4 (and/or A5–B5), B12 and C1. The fenitrothion resistant populations were highly resistant to propoxur indicating the possible involvement of their common target site (AChE 1) in the recorded resistance. Our results are particularly interesting to update the information base for rational deployment of the existing tools for mosquito's control.