This article describes the formation of crosslinked 10-200-nm-thick polymer hydrogel films by alternating the spin-coating of two mutually reactive polymers from organic solutions, followed by hydrolysis of the resulting multilayer film in aqueous buffer. Poly(methyl vinyl ether-alt-maleic anhydride) (PMM) was deposited from acetonitrile solution, and poly(N-3-aminopropylmethacrylamide-co-N-2-hydroxypropylmethacrylamide) (PAPM(x), where x corresponds to the 3-aminopropylmethacrylamide content ranging from 10 to 100%) was deposited from methanol. Multilayer films were formed in up to 20 deposition cycles. The films cross-linked during formation by reaction between the amine groups of PAPMx and the anhydride groups of PMM. The resulting multilayer films were covalently postfunctionalized by exposure to fluoresceinamine, decylamine, D-glucamine, or fluorescently labeled PAPM(x) solutions prior to the hydrolysis of residual anhydride in aqueous PBS buffer. This allowed tuning the hydrophobicity of the film to give static water contact angles ranging from about 5 to 90 degrees. Increasing the APM content in PAPM(x) from 10 to 100% led to apparent Young's moduli from 300 to 700 kPa while retaining sufficient anhydride groups to allow postfunctionalization of the films. This allowed the resulting (PMM/PAPM(x)) multilayer films to be turned into adhesion-promoting or antifouling surfaces for C2C12 mouse myoblasts and MCF 10A premalignant human mammary epithelial cells.