Probiotics are effective to treat or prevent gastrointestinal infections, and microalgae have demonstrated important health-promoting effects and in some cases function as prebiotics. In this regard, the anti-rotavirus effect ofBifidobacterium longumandChlorella sorokinianaby reducing viral infectivity is well known. However, their effect on immune response against rotavirus has not yet been investigated. Therefore, the aim of this study was to determine the role ofBifidobacterium longumand/orChlorella sorokinianain influencing an IFN type I-mediated antiviral response in rotavirus-infected cells. In pre-infection experiments, HT-29 cells were treated withB. longumandC. sorokinianaalone or in combination, followed by rotavirus infection, whereas in post-infection assays, HT-29 cells were treated after infection. The cells’ mRNA was then purified to determine the relative expression level of IFN-α, IFN-β, and precursors of interferons such as RIG-I, IRF-3, and IRF-5 by qPCR. We showed that combination ofB. longumandC. sorokinianasignificantly increased IFN-α levels in pre-infection and IFN-β in post-infection assays, as compared with individual effects. Results indicate thatB. longum,C. sorokiniana, or their combination improve cellular antiviral immune response.