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The ecological consequences of mosquito control using the larvicide Bacillus thuringiensis israelensis (Bti) are still a matter of debate especially when it comes to adverse effects on non-target but susceptible non-biting midges (Chironomidae). Our work aimed to assess the effects of Bti applied in operational mosquito control rates on chironomid abundances in seasonal flooded freshwater wetlands. We assessed the invertebrate community alongside with aquatic insect emergence rates in studies with increasing ecotoxicological complexity, ranging from artificial mesocosms, over a semi-field approach using enclosures to natural conditions in field studies. Study sites represented different habitats (forest, meadow, floodplain) with regularly Bti treated and untreated temporal wetlands that are located in the Upper Rhine Valley, Germany. Larval chironomids were the most affected group of organism in the altered aquatic invertebrate communities of the Bti treated pond mesocosms. The larvae of the chironomid subfamilies Chironominae and Orthocladiinae were affected by Bti field concentrations while emergence rates of Tanypodinae did not change. The Bti treatment significantly reduced the targeted mosquitoes in the mesocosm and in the field studies. At the same time, however, the application of operational Bti field rates reduced overall chironomid emergence rates to about half of the control rates. The observed abundance reductions occurred independently of the ecological complexity in the study design in artificial mesocosms as well as realistic field conditions. The considerable reduction of the abundant non-target chironomids along with mosquitoes may subsequently lead to unwanted indirect negative effects for birds, bats and other aquatic organisms feeding on midges. Hence, large-scale applications of Bti for mosquito control in seasonal wetlands should be considered more carefully. This is of special importance when these wetlands are parts of national parks, nature reserves or Natura 2000 sites that were created for the protection of nature and environmental health., (Copyright © 2018 Elsevier Inc. All rights reserved.)