Herein, we report the design and calculated molecular properties of ten existing azo Schiff bases 5a–h and 7a, b. On the basis of a hypothetical antibacterial pharmacophore, the structures are designed to interact with Gram-positive and Gram-negative bacteria. The in vitro biological evaluation of these compounds allowed us to point out new potential non-nucleoside hits, with MIC values in the range of 2–8 lg/ml active against Staphylococcus aureus and Bacillus subtilis. In contrast to Gram positive strains, no activity is noted in favor of Gram negative strains (Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli). The antibacterial activity of these azo Schiff bases is discussed on the basis of theoretical calculations by using Petra, Osiris and Molinspiration (POM) model.