By repurposing the recently popularized expansion microscopy to control the meshwork size of hydrogels, we examine the size-dependent suppression of molecular diffusivity in the resultant tuned hydrogel nanomatrices over a wide range of polymer fractions of ∼0.14-7 wt %. With our recently developed single-molecule displacement/diffusivity mapping (SMdM) microscopy methods, we thus show that with a fixed meshwork size, larger molecules exhibit more impeded diffusion and that, for the same molecule, diffusion is progressively more suppressed as the meshwork size is reduced; this effect is more prominent for the larger molecules. Moreover, we show that the meshwork-induced obstruction of diffusion is uncoupled from the suppression of diffusion due to increased solution viscosities. Thus, the two mechanisms, respectively, being diffuser-size-dependent and independent, may separately scale down molecular diffusivity to produce the final diffusion slowdown in complex systems like the cell.