The aim of this study was to evaluate the susceptibility of Anopheles gambiae mosquitoes to bendiocarb, before (2010) and after (2012) the implementation of indoor residual spraying (IRS) interventions and to report the evolution of Ace-1R mutation frequency in Atacora region. Indoor collection was carried out through Morning 7 to 9 a. m in five districts (Kouandé, Natitingou, Matéri, Tanguiéta and Copargo) of the Atacora-Donga region before and after IRS. Anopheles larvae were also reared in each district before and after IRS and emerging adults were exposed to bendiocarb (0.1%) in susceptibility tests. Polymerase chain reaction (PCR) assays were run to determine the members of the An. gambiae complex, as well for insensitive acetylcholinesterase (AChE1) due to Ace-1R mutation. This study showed that the mean Ace-1 mutation frequency had increased significantly from 2010 to 2012 after two years of an IRS. Mortality data indicated that mosquitoes were susceptible in 2010 to bendiocarb 0.1%. By 2012, there was a drastic decline in the An. gambiae susceptibility to bendiocarb in treated districts. An. gambiae s.s. and Anopheles coluzzi were the two members of An. gambiae complex that were found in sympatry in the study area. An. gambiae was predominant in tested samples (92.50%). The Ace-1R mutation was found in both An. gambiae s.s. and An. coluzzi with frequency of 7.33 and 7.35%, respectively. The high proportion of homozygous susceptible specimens that survived from the WHO bioassays suggests the implication of biochemical resistance mechanisms. These results are of prime importance in the effort to document multiple impacts of operational control programmes on mosquito vectors, and to conceive sustainable control strategies for future malaria control programmes