Cellular environments such as nanoconfinement and molecular crowding can change biomolecular properties. However, in nanoconfinement, it is extremely challenging to investigate effects of crowding cosolutes on macromolecules. By using optical tweezers, here, we elucidated the effects of hexaethylene glycol (HEG) on the mechanical stability of a telomeric G-quadruplex (GQ) in a zeptoliter DNA origami reactor (zepto-reactor). When HEG molecules were introduced in the GQ-containing zepto-reactor at different positions, we found that the GQ species split into two equilibrated populations, reflecting diverse effects of the oligoethylene glycol on the GQ via either a long-range dehydration effect or direct interactions. When the number of HEG molecules was increased, the stability of the GQ unexpectedly decreased, suggesting that the direct destabilizing interaction between the GQ and HEG is dominating over the long-range stabilizing dehydration effects of the HEG in hydrophilic nanocavities. These findings indicate that a nanoconfined environment can alter regular effects of cosolutes on biomacromolecules.