Freestanding films based on catechol functionalized chitosan (CHI), hyaluronic acid (HA), and bioglass nanoparticles (BGNPs) were developed by spin-coating layer-by-layer assembly (SA-LbL). The catechol groups of 3,4-dihydroxy-l-phenylalanine (DOPA) present in the marine mussels adhesive proteins (MAPs) are the main factors responsible for their characteristic strong wet adhesion. Then, the produced films were cross-linked with genipin to improve their stability in wet state. Overall, the incorporation of BGNPs resulted in thicker and bioactive films, hydrophilic and rougher surfaces, reduced swelling, higher weight loss, and lower stiness. The incorporation of catechol groups onto the films showed a significant increase in the films’ adhesion and stiness, lower swelling, and weight loss. Interestingly, a synergetic eect on the stiness increase was observed upon the combined incorporation of BGNPs with catechol-modified polymers, given that such films were the stiest. Regarding the biological assays, the films exhibited no negative eects on cellular viability, adhesion, and proliferation, and the BGNPs seemed to promote higher cellular metabolic activity. These bioactive LbL freestanding films combine enhanced adhesion with improved mechanical properties and could find applications in the biomedical field, such as guided hard tissue regeneration membranes.