The literature reports that more research needs to be done on using chitosan as an environmentally friendly bioinsecticide, especially against dipterans. Thus, we examined the effect of chitosan when fed, in no-choice experiments, to adult Musca domestica, Tabanus nigrovittatus, and Phormia regina. A 2% chitosan solution was fed, in no-choice experiments, to adults of Musca domestica, Tabanus nigrovittatus, and Phormia regina to study the effects on survivorship. In all species, the uptake of chitosan caused a significant decrease in survivorship. Examination of the digestive tract of house flies showed a thick material within the midgut, plus a shriveling of the midgut only in chitosan-treated flies. A survivorship curve of adult house flies fed a 10% sucrose and 2% w/v ascorbic acid-only solution showed that the effect of the chitosan solution was due to the chitosan and not the ascorbic acid. Intake experiments revealed that by day 2, chitosan treated house flies consumed significantly less of the diet compared to controls. Chitosan, a biopesticide, when fed in no-choice experiments to three species of adult flies produced severe mortality within 4-6 days of ingestion. A working hypothesis suggests that by day 2 of the chitosan diet there appears to be a malfunction of the digestive tract and possibly the midgut microbiome. © 2020 Society of Chemical Industry.