Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA-membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The Kd values range from 8.5 ± 4.9 to 466 ± 134 μM whereby negatively charged headgroups led to weak binding due to the electrostatic repulsion with respect to the negatively charged DNA. Atomistic MD simulations explain the findings and elucidate the dynamic nature of anchored DNA such as the mushroom-like conformation of single-stranded DNA hovering over the bilayer surface in contrast to a straight-up conformation of double-stranded DNA. The biophysical insight into the binding strength to membranes as well as the molecular accessibility of DNA for hybridization to molecular cargo is expected to facilitate the creation of biomimetic DNA versions of natural membrane nanopores and cytoskeletons for research and nanobiotechnology.