Microalgae are proposed in several biotechnological fields because of their ability to produce biomass enriched in high-value compounds according to cultivation conditions. Regarding the health sector, an emerging area focuses on natural products exploitable against viruses. This work deals with the characterization of the green microalga Neochloris oleoabundans cultivated under autotrophic and mixotrophic conditions as a source of whole aqueous extracts, tested as antivirals against HCoV-229E (Coronaviridae family). Glucose was employed for mixotrophic cultures. Growth and maximum quantum yield of photosystem II were monitored for both cultivations. Algae extracts for antiviral tests were prepared using cultures harvested at the early stationary phase of growth. Biochemical and morphological analyses of algae indicated a different content of the most important classes of bioactive compounds with antiviral properties (lipids, exo-polysaccharides, and total phenolics, proteins and pigments). To clarify which phase of HCoV-229E infection on MRC-5 fibroblast cells was affected by N. oleoabundans extracts, four conditions were tested. Extracts gave excellent results, mainly against the first steps of virus infection. Notwithstanding the biochemical profile of algae/extracts deserves further investigation, the antiviral effect may have been mainly promoted by the combination of proteins/pigments/phenolics for the extract derived from autotrophic cultures and of proteins/acidic exo-polysaccharides/lipids in the case of mixotrophic ones.