Films of agarose gel microspotted with polylysine aqueous solution have been characterized by atomic force microscopy carried out in deionized water. Thickness and surface morphology of the layers have been checked, and the effect of polylysine impregnation on the local elasticity has been investigated. An increase in contact stiffness of the organic layer at the spotted areas has been observed, correlated with the polylysine concentration. For the considered agarose layer thickness of ~0.9 μm in dry condition, the concentration threshold at which stiffening appears is ~0.1 mg/mL. Above this threshold, the stiffening coefficient becomes approximately twofold and seems not to increase significantly with concentration in the range 0.3-0.7 mg/mL. For concentrations above the stiffening threshold, this effect is also accompanied by a locally lower film thickness. For quantitative determination of the stiffness, force-distance curves extracted from the regions of interest of spots and agarose substrate have been selected and processed. These curves were fitted to the Hertz model of purely elastic tip-surface interaction, under appropriate assumptions on both tip shape and optimum indentation depth. In this way, we could determine the Young's modulus of the agarose layer to be ~50 kPa and quantitatively confirm the stiffening due to polylysine.