Structurally diverse 1-aryl-10H-[1,2,4]triazolo[30 ,40 :3,4][1,2,4]triazino[5,6-b]indoles 4a–v were synthesized by regiospecific heterocyclizations. The designed molecular diversity was evaluated in vitro in parallel cellbased assays for cytotoxicity of viruses multiplication supporting cell lines and antiviral activity against viruses representative of two of three genera of the Flaviviridae family. The compound library was also tested against Retrovirus (HIV-1), two Picornaviruses (CVB-2 and Sb-1), and Paramyxoviridae (VSV) representative. Among double-stranded RNA (dsRNA) viruses, Reoviridae representative (Reo-1) was tested. Two representatives of DNA virus families were also included—HSV-1 (Herpesviridae) and VV (Poxviridae). The compounds 4m and 4o were found cytotoxic, having CC50 values ranging from 4 to 30 lM. Moreover, compound 4v has exhibited significant activity (EC50 = 3 lM) against BVDV.