The expanded uses of bioplastics require understanding the potential health risks associated with their exposure. To address this issue, Drosophila melanogaster as a versatile terrestrial in vivo model was employed, and polylactic acid nanoplastics (PLA-NPLs), as a proxy for bioplastics, were tested as a material model. Effects were determined in larvae exposed for 4 days to different concentrations (25, 100, and 400 μg/mL) of 463.9 ± 129.4 nm PLA-NPLs. Transmission electron microscopy (TEM) and scanning electron microscope (SEM) approaches permitted the detection of PLA-NPLs in the midgut lumen of Drosophila larvae, interacting with symbiotic bacteria. Enzymatic vacuoles were observed as carriers, collecting PLA-NPLs and enabling the crossing of the peritrophic membrane, finally internalizing into enterocytes. Although no toxic effects were observed in egg-to-adult survival, cell uptake of PLA-NPLs causes cytological disturbances and the formation of large vacuoles. The translocation across the intestinal barrier was demonstrated by their presence in the hemolymph. PLA-NPL exposure triggered intestinal damage, oxidative stress, DNA damage, and inflammation responses, as evaluated via a wide set of marker genes. Collectively, these structural and molecular interferences caused by PLA-NPLs generated high levels of oxidative stress and DNA damage in the hemocytes of Drosophila larvae. The observed effects point out the need for further studies aiming to deepen the health risks of bioplastics before adopting their uses as a safe plastic alternative.