The aim of the present study was to characterise anti-vaccinia virus activities of anionic exopolysaccharides TK V3 isolated from cyanobacterium Arthrospira platensis and an exopolysaccharide isolated from the rhodophyt Porphyridium purpureum, respectively. These substances have previously been shown to be active against other enveloped viruses. We determined the in vitro inhibition of GFP-expressing vaccinia virus replication of 50% at a concentration of 0.65 µg/ ml for EPS and of 0.78 µg/ml for TK V3. Substances also had an antiviral effect against ectromelia virus which is a most distinct orthopoxvirus genetically and the causative agent of mousepox. Anti-vaccinia virus and anti-ectromelia virus activities were demonstrated to increase with decreasing multiplicity of infection. Furthermore, non-toxic reduction of vaccinia virus in ovo replication was shown. Using time-of-addition assays, inhibition of viral entry by polyanionic substances was verified. EPS and TK V3 derived from phototrophic microorganisms represent novel anti-orthopoxvirus substances.