Polyelectrolyte multilayers (PEMs) are now widely used for biomedical applications. In this work, we investigated the primary osteoblast adhesion properties of PEMs of poly(L-lysine) (PLL), poly(L-glutamic acid) (PGA), poly(alginic acid) (Palg), and poly(galacturonic acid) (Pgal). In order to compensate for the poor adhesion of the as-synthesized films, two kinds of film modifications were achieved: a purely physical modification by film crosslinking, and a chemical modification by grafting a arginine–glycine–aspartic acid (RGD) peptide to PGA. Crosslinking was performed using a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide (sulfo-NHS) to induce amide formation. This reaction was followed by Fourier-transform IR spectroscopy. For film functionalization, a 15-amino-acid peptide was grafted to PGA and deposited as the top layer of the film. PLL/PGA, PLL/Palg, and PLL/Pgal films were crosslinked or functionalized. The films were tested for both short-term adhesion properties and long-term proliferation of primary osteoblasts. Whereas the effect of film crosslinking on short-term adhesion was moderate, it was much more important for the RGD-functionalized films. On the other hand, the long-term proliferation was the same or even higher for the crosslinked films as compared with the functionalized films. This effect was particularly enhanced for the PLL/Palg and PLL/Pgal films. Finally, we functionalized PLL/PGA that had been crosslinked prior to PGA-RGD deposition. These architectures exhibited even higher short-term adhesion and proliferation. These results clearly show the important role of the physical properties of the films, besides their chemical properties, for the modulation of primary cell-adhesion behavior.