The preparation of alginate films with suitable properties requires a two-step contact with reticulating agents: initially a weakly structured pre-film is formatted which is further crosslinked in a second stage by immersion in a more concentrated solution. The present work evaluated the effects of a combined crosslinking procedure using calcium and barium ions on the physical and morphological properties of alginate-based films containing natamycin as antimicrobial agent. The release behavior of natamycin in water was evaluated as well as the antimicrobial activity against four target microorganisms, which are common cheese product contaminants. Films attributes were affected by the type of ion used in the second stage while the natamycin release rate and the antimicrobial activity were influenced by the ion used in the first stage. Films crosslinked with Ba2+ in the first and Ca2+ in the second stage (Ba–Ca films) exhibited physical properties very similar to films crosslinked with calcium in both stages. Release kinetics of natamycin in water fitted well to Fick's second law diffusional model, with effective diffusivity values ranging from 0.40 × 10−11 to 1.74 × 10−11 cm2/s. Ba–Ca films presented the lowest natamycin diffusion coefficient and the smallest inhibition zone diameter against the four microorganisms tested Ver menos