A simple, rapid and one-pot quadruple Mannich reaction has been developed for the synthesis of 2,5,7,9,11-pentaazaphenalenes via a three-component reaction of 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one, formaldehyde and primary amines in ethanol at room temperature. The reaction products were obtained in very good to excellent yields using a simple work-up procedure. The regioselectivity of the reaction was studied using the electronic energy calculations which indicated that the corresponding products 2,5,7,9,11-pentaazaphenalenes are more favorable than the other isomeric products 3,5,8,10,11-pentaazaanthracenes and 2,5,7,8,10-pentazaphenanthrenes. The reaction mechanism was investigated and the structures of all the new compounds were confirmed using spectra and elemental analysis. In addition, all the synthesized compounds were screened for antimicrobial activity and showed a significant activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Geotrichum candidum and Trichophyton rubrum.