Cellulose films of nanometric thickness were produced by spin-coating on GaAs(100). Films were deposited using cellulose silylated solutions and subsequently regenerated by exposing them to vapors of hydrochloric acid. After regeneration, these films can strongly resist solvents. Modification of the film surface region was performed by immersing the regenerated cellulose films in a solution of phenyl isocyanate in dimethyl sulfoxide. A different functionalization was also successfully achieved through the interaction of the film surface with 4,4′-methylenebis(phenyl isocyanate) (MDI). Surfaces treated with MDI keep an unreacted isocyanate group and can again be modified by amines. For this purpose, 4-bromoaniline was used. All kinetics of the different molecular interactions with the cellulose film on GaAs were followed in situ using FTIRS in ATR/MIR (attenuated total reflection in multiple internal reflections) mode. Besides ATR/MIR having an analysis depth on the order of 1 µm, other surface techniques were used for analyzing these films with other probing depths such as X-ray photoelectron spectroscopy with ∼10 nm and high-resolution electron energy loss spectroscopy with ∼1 nm in the impact regime. The set of methods presented here represents a quite adequate way to study the surface chemistry of cellulose films and the procedures for their functionalization