A computational model has been developed for the rational design of bioactive pharmacophore sites as an antibacterial, antifungal and antiviral candidates based on available X-ray structures of bis-Schiff bases (Blagus et al., Maced J Chem Chem Eng 29:117–138, 2010; Nabei et al., Polyhedron 28:1734–1739, 2009; Zhang et al., Inorg Chem Commun 14:1636–1639, 2011; Zhong et al., Eur J Med Chem 41:1090–1092, 2006; Zhou et al., Inorg Chim Acta 359:1442–1448, 2006). A dozen of bis-Schiff bases 3–14 of isatin, benzylisatin and 5-fluoroisatin 1a–c were designed using this model. The compounds were screened for antibacterial, antifugal and antiviral activity against a panel of DNA and RNA viruses. The most potent of these compounds 8 and 11 was tested in viral cultures for their ability to present a potential (Od-–Nd-) antiviral pharmacophore site. Compounds 8 and 11 were the most cytotoxic in HEL cells. All these synthesized bis-Schiff bases were also tested for their antibacterial and antifungal activities. They did not display activity against S. cerevisiae (ATCC 28383) or C. albicans (CIP 1180-79); may be because they did not have an antibacterial pharmacophore site (Xd-–Yd?). The best inhibitors tested in vitro against HIV-1 are genetically predisposed to be inhibited by similar pharmacophore sites. The results from all the aspects of this bioinformatic approaches are discussed as par with our experience with screening candidates.