Diflubenzuron (DFB, trade name dimilin 25 WP) is a chitin synthesis inhibitor widely used against forest insect pests in Algeria. Prior to implementation of these products as chemical agents for mosquito control, knowledge on their potential effects on non-target organisms and its behaviour in water are needed. Therefore, the present study was focused on DFB and aimed to use an HPLC procedure developed previously in order to obtain information on its degradation in freshwater and its bioconcentration in adult females of the fish Gambusia affinis (Cyprinodondiformes, Poeciliidae), which is one of the best candidates for biological control programs against mosquitoes. The adult females were exposed to dimilin (initial concentration 312 ng a.i./ml) for 28 days and residues analysis determined at different exposure times (0, 7, 14, 21 and 28 days). The concentration of DFB in freshwater decreased with exposure time while the amount of residues detected at the surface of the fish body increased progressively to reach a maximum at day 14 (162.7 ± 14.0 ng/fish) and declined thereafter during the exposure period. In addition, DFB incorporation into body increased with decreasing DFB concentration in water at each exposure time. The following average distribution was noted at the end of experiment (28 days): about 33% of the applied concentration was detected on the surface of fish body and was recovered by simple rinsing, and about 67% was found inside the fish body. A degradation in water and surface of fish occurred starting day 14 during the experimental period. Thus, about 40% of the initial concentration was degraded in freshwater after 28 days. The results are discussed to develop a better understanding of the degradation of dimilin in water and their potential effect on non-target organisms for its application for controlling mosquito.