The low gene transfer efficiency of chitosan-DNA polyplexes is a consequence of their high stability and consequent slow DNA release. The incorporation of an anionic polymer is believed to loosen chitosan interactions with DNA and thus promote higher transfection efficiencies. In this work, several formulations of chitosan-DNA polyplexes incorporating hyaluronic acid were prepared and characterized for their gene transfection efficiency on both HEK293 and retinal pigment epithelial cells. The different polyplex formulations showed morphology, size, and charge compatible with a role in gene delivery. The incorporation of hyaluronic acid rendered the formulations less stable, as was the goal, but it did not affect the loading and protection of the DNA. Compared with chitosan alone, the transfection efficiency had a 4-fold improvement, which was attributed to the presence of hyaluronic acid. Overall, our hybrid chitosan-hyaluronic acid polyplexes showed a significant improvement of the efficiency of chitosan-based nonviral vectors in vitro, suggesting that this strategy can further improve the transfection efficiency of nonviral vectors.