<p>Hydrogel films were prepared <i>via in situ</i> physical cross-linking of guanidinium polyampholytes and kappa-carrageenan by sodium tetraborate and potassium chloride, respectively. Composite films loaded with doxycycline were prepared according to the “equilibrium” (saturation) method. The kinetics for the doxycycline release from these hydrogels was most successfully described within the framework of Higuchi model, which indicates that the antibiotic release is the diffusion-controlled process. The best linear fit according to the values of the determination coefficient <i>r</i>² for the doxycycline release from the hydrogel based on carrageenan and copolymer of 2,2-diallyl-1,1,3,3-tetraethylguanidine chloride and acrylic acid was achieved within the framework of Korsmeyer—Peppas model. Values of the release degree n within the range of 0.553–0.633 indicate a “non-Fickian” diffusion mechanism, which might be due to the relaxation of polymer chain. It was found that our doxycycline-loaded films exhibit a pronounced antibacterial effect.</p>