Chitosan/biomimetic apatite thin films were grown in mild conditions of temperature and pressure by Combinatorial Matrix-Assisted Pulsed Laser Evaporation on Ti, Si or glass substrates. Compositional gradients were obtained by simultaneous laser vaporization of the two distinct material targets. A KrF* excimer (λ=248nm, τFWHM=25ns) laser source was used in all experiments. The nature and surface composition of deposited materials and the spatial distribution of constituents were studied by SEM, EDS, AFM, GIXRD, FTIR, micro-Raman, and XPS. The antimicrobial efficiency of the chitosan/biomimetic apatite layers against Staphylococcus aureus and Escherichia coli strains was interrogated by viable cell count assay. The obtained thin films were XRD amorphous and exhibited a morphology characteristic to the laser deposited structures composed of nanometric round shaped grains. The surface roughness has progressively increased with chitosan concentration. FTIR, EDS and XPS analyses indicated that the composition of the BmAp-CHT C-MAPLE composite films gradually modified from pure apatite to chitosan. The bioevaluation tests indicated that S. aureus biofilm is more susceptible to the action of chitosan-rich areas of the films, whilst the E. coli biofilm proved more sensible to areas containing less chitosan. The best compromise should therefore go, in our opinion, to zones with intermediate-to-high chitosan concentration which can assure a large spectrum of antimicrobial protection concomitantly with a significant enhancement of osseointegration, favored by the presence of biomimetic hydroxyapatite.