Over the past decade, much attention has been paid to chitosan as a potential drug carrier because of its non-toxicity, biocompatibility, biodegradability and antibacterial properties. The effect of various chitosan characteristics on its ability to carry different antibiotics is discussed in the literature. In this work, we evaluated the influence of the different molecular weights of this polymer on its potential as an antibacterial membrane after adding gentamicin (1%w/w). Three types of chitosan membranes without and with antibiotic were prepared using a solvent casting process. Their microstructures were analyzed with a 4K digital microscope, and their chemical bonds were studied using FTIR spectroscopy. Furthermore, cytocompatibility on human osteoblasts and fibroblasts as well as antibacterial activity againstStaphylococcus aureus(S. aureus.) andEscherichia coli(E. coli) were assessed. We observed that the membrane prepared from medium-molecular-weight chitosan exhibited the highest contact angle (≈85°) and roughness (10.96 ± 0.21 µm) values, and its antibacterial activity was unfavorable. The maximum tensile strength and Young’s modulus of membranes improved and elongation decreased with an increase in the molecular weight of chitosan. Membranes prepared with high-molecular-weight chitosan possessed the best antibacterial activity, but mainly againstS. aureus. ForE. coli, is not advisable to add gentamicin to the chitosan membrane, or it is suggested to deplete its content. None of the fabricated membranes exhibited a full cytotoxic effect on osteoblastic and fibroblast cells. Based on our results, the most favorable membrane as a gentamicin carrier was obtained from high-molecular-weight chitosan.