Layer-by-layer multilayer films of poly(styrenesulfonate) (PSS) and 4th generation poly(amidoamine) dendrimer (4G PAMAM) assembled on particle supports were modified by a stepwise process involving (i) cross-linking of free amino groups of PSS/4G PAMAM multilayers using a bifunctional cross-linking agent, gluteraldehyde, which simultaneously chemically activates the surface, (ii) covalently linking the free aldehyde groups made available from the cross-linker with octadecylamine (fatty-acylation), and (iii) adsorbing a poly(ethylene glycol) (molecular weight similar to2000) derivative of distearylphosphatidylethanolamine (PEG2000-DSPE) on the fatty-acylated surface (PEGylation). Microelectrophoresis, confocal laser scanning microscopy, transmission electron microscopy, and differential scanning calorimetry were used to follow the modification of the PSS/4G PAMAM multilayer coatings on the particles. Analogous experiments were performed on planar supports (silicon, glass, and gold) to additionally quantify the process by Fourier transform infrared spectroscopy, atomic force microscopy, contact-angle, ellipsometry, and quartz crystal microbalance techniques. The experimental data from the colloid and planar supports were in mutual agreement, providing evidence for the stepwise surface modification of the multilayer films. Adsorption experiments involving biological cells or proteins and the surface-modified PSS/4G PAMAM multilayers were conducted to establish the surface biocompatibility of the modified films. Compared with the respective unmodified films, the surface-modified PSS/4G PAMAM multilayer-coated colloid particles showed reduced adhesion to the biological cell surfaces (macrophage cell line TPH-1), while the adsorbed amount of human serum albumin decreased upon exposure to the surface-modified PSS/4G PAMAM multilayer-coated planar substrates. This demonstrates the biocompatibility of the surface-modified multilayer films.