Using cotton cultivars that express a gene of the Bacillus thuringiensis (Bt) bacterium producing a protein (Cry1Ac) with an insecticide effect on the Lepidoptera pests has made it possible to reduce the number of insecticide applications during the crop cycle. Thus, the objective was to determine, in the field during the 2006/2007 harvest in Dourados/MS, Brazil, the impact of the transgenic cultivar (NuOpal(R)) by comparison with the isogenic, non-transgenic cultivar (DeltaOpal(R)) on target pests, non-target pests and natural enemies using two sampling methods (beatsheet and whole plant observation) under conventional growing conditions, with both varieties cultivated in a system incorporating the application of insecticides for non-target pests that reached the recommended threshold level for integrated pest management. It was verified that the average number of target pest specimens for both sampling methods was significantly lower in Bt-cotton than in non-Bt-cotton. However, the average number of non-target pest specimens and natural enemies presented no significant differences between the cultivars for both sampling methods assessed. The diversity of non-target pests characterized by the Shannon-Wiener index presented a significant difference between Bt-cotton and non-Bt-cotton for the whole plant sampling method, whereas for naturally occurring enemies, no difference was revealed using this sampling method.