This study aimed to analyze the possible effects of diflubenzuron on the associated insect fauna under laboratory, semi-field and field conditions. Laboratory bioassays were performed in aquariums with mortality readings (%) every 24h until 96h, and in semi-fields, insects were kept in basins with readings every 24h for up to 12 days, in triplicates and a control. In the field experiment, a collection was performed before the application of diflubenzuron in ten brick factory pits (25m²) and 15 post-application. The values of LC 50 and LC 90 for Chironomus were 2.77x10-3g/L and 0.86g/L, respectively, and for Buenoa, they were 0.019g/L and 0.92g/L, a strong relationship was observed between mortality and exposure time (r²>0.8) in all concentrations used. In semi-field, similar mean values of emergency inhibition were observed, except for Euthyplociidae, which was more sensitive. There was no significant difference between the data of richness and diversity of aquatic insects in the field experiment, considering an interval of 15 days (p=0.32). Finally, the data suggest that diflubenzuron may have a negative effect on the associated insect fauna, but in the field experiment the environmental conditions of Anopheles breeding sites may have affected its efficiency.