Environmental and health issues resulting from the intensive use of broad-spectrum chemical insecticides, have favored the spread of biological insecticides in the last decades. The most common, Bacillus thuringiensis (Bt), is a spore-forming bacterium which produces a varied range of insecticidal toxins. However, the field accumulation of spores and toxins of Bt kurstaki (Btk), the Bt subspecies most commonly used against lepidopteran pests, potentially increases the risk for non-target biodiversity. Using mainly the Lepidoptera-targeting Bt subspecies kurstaki (Btk) and the well-studied laboratory models Drosophila spp. as non-target insects, we tested the potential non-intentional effects of the ingestion of commercial Btk formulations on traits related to the fly development and the adult fitness. We observed a delayed and reduced emergence of Drosophila melanogaster flies at doses above 107 CFU/g of food (close to the doses used for fields treatments), regardless of the fly strain, the commercial formulation, and the rearing medium. Mortality affected mainly the early larval stage while all the larvae died before pupation at the highest dose (109 CFU/g). We analyzed the respective roles of the formulation components (spores, toxins, additives…), and found that the developmental and lethal effects may result from a synergy between Btk spores and toxins. Developmental and lethal impacts were also observed for other Drosophila species, with variation in the LD50 and in the effect amplitude. Our work suggests that the environmental accumulation of Bt may have possible non-intentional effects on non-target Drosophila species and the associated species communities.