This study investigates the impact of different Atomic Layer Deposition techniques on the Ti-metalation of chitosan (CS) for biomedical applications. Three methods – vapour phase metalation (VPM), multiplied pulsed vapour phase infiltration (MPI), and O₂ Plasma Enhanced ALD (PEALD) – were employed using Tetrakis(dimethylamino)titanium as the precursor. X-ray photoelectron spectroscopy revealed varying degrees of titanium chemisorption and Ti–O bond formation, highlighting the role of water in influencing the structure and properties of the resulting Ti–CS films. X-ray diffraction analysis showed significant structural changes depending on the deposition method. VPM reduced crystallite size and overall crystallinity, particularly in hydrated and anhydrous CS phases. In contrast, MPI and PEALD treatments favoured growth in the regular crystalline phase, with MPI showing the greatest enhancement, indicating a stronger phase transformation induced by water. These structural variations directly influenced the films’ semiconductor behaviour, as evidenced by changes in optical band gaps across the samples. Contact angle and surface energy analyses revealed increased hydrophobicity and lower water affinity in MPI films, while PEALD-treated surfaces exhibited the highest wettability and apparent surface energy, attributed to Ti–OH enrichment and plasma-induced rehydration. Water was used as the probe liquid due to its biomedical relevance and sensitivity to surface polarity. The Ti–CS films exhibited semiconductor characteristics, with varying optical band gaps observed. PEALD films demonstrated the highest C2C12 cell confluence, attributed to improved wettability, conductivity, and purity of Ti species. Antibacterial activity was observed across all Ti–CS films, with VPM exhibiting the highest efficacy against Helicobacter pylori and Escherichia coli . These findings highlight the potential of tailoring Ti–CS film properties through ALD techniques for various medical applications, including antiseptic coatings, tissue engineering scaffolds, and intelligent implants.