Here we showed that aqueous extract of alligator plant, Bryophyllum daigremontianum L., is able to inhibit systemic and local infection of Tobacco mosaic virus (TMV: family Virgaviridae) in broad bean, Vicia faba L., nettle-leaved goosefoot, Chenopodium murale L., and tobacco, Nicotiana tabacum var. Turkish, N. tabacum var. Xanthi and N. glutinosa L., hosts. Antiviral activity of the extract was retained for a period of 8 days on treated broad bean and tobacco leaves. This activity was negatively correlated with the extract concentration, and it was completely lost by washing the treated leaves of tobacco plants 2 h post application, and was ineffective when applied 24 h post inoculation. No inhibitory effect was found against agro-inoculated strain of Beet curly top virus (BCTV: family Geminiviridae) on sugarbeet, Beta vulgaris L., seedlings. To determine the antiviral agent, Bryophyllum bulk protein designated BBP was isolated from the extract. BBP exhibited RNase activity against total RNA of TMVinfected tobacco tissues and genomic RNA of TMV while it failed to degrade genomic DNA of BCTV. Additionally, BBP completely inhibited TMV on N. glutinosa leaves at concentration of 40 μ g/ml. These results suggest that a ribonuclease is mainly responsible for antiviral activity of alligator plant extract. To our knowledge, this is the first report on inhibitory effect of alligator plant extract on a plant virus. This plant species can be considered as a promising source for antiviral proteins in order to develop plant-derived compounds for effective control of plant mosaic diseases caused by TMV.