In this study, we develop chitosan–hydroxyapatite (CS–HAp) composite layers that were deposited on Si substrates in radio frequency (RF) magnetron sputtering discharge in argon gas. The composition and structure of CS–HAp composite layers were investigated by analytical techniques, such as Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), metallographic microscopy (MM), and atomic force microscopy (AFM). On the other hand, in the present study the second order derivative of FT-IR–ATR spectra, for compositional analyses of CS–HAp, were used. The SEM, MM, and AFM data have shown the formation of CS–HAp composite layers. The surface of CS–HAp composite layers showed uniform growth (at an Ar gas working pressure of p = 2 × 10−3 mbar). The surface of the CS–HAp composites coatings became more nanostructured, becoming granular as the gas pressure increased from 5 × 10−3 to 1.2 × 10−2 mbar. However, our studies revealed that the surface morphology of the CS–HAp composite layers varies with the Ar gas working pressure. At the same time, optical properties are slightly influenced by Ar pressure. Their unique physicochemical properties make them suitable for various applications in the biomedical field, if we consider the already proven antimicrobial properties of chitosan. The antifungal properties and the capacity of the CS–HAp composite layers to inhibit the development of fungal biofilms were also demonstrated using the Candida albicans ATCC 10231 (C. albicans) fungal strain.