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Acetic acid, a colourless liquid organic acid with a characteristic acrid smell, is obtained naturally and has applications in both the food and pharmaceutical industries. It has been reported to have beneficial uses for lifestyle-related diseases, and its efficient disinfectant properties are well known. In this study, an alginate crosslinked with Ca²⁺ hydrogel film was treated with acetic acid to explore its biological properties for biomedicine. The results showed that the novel calcium alginate/acetic acid film was biocompatible in vitro using human keratinocyte cells and in vivo with <i>Caenorhabditis elegans</i>. It also had antiviral properties against enveloped and non-enveloped viruses and anticancer properties against melanoma and colon cancer cells. This novel film thus showed promise for the biomedical and pharmaceutical industries, with applications for fabricating broad-spectrum antiviral and anticancer materials.