In spite of phenobarbital has been used in various medical fields as hypnotics, anxiolytics, and anticonvulsants, it also contains active functional groups that can be reacted to form other products as dyes, polymers, antimicrobial and anti-antioxidants agents. A series of barbituric acid derivatives containing 1,2,3,4-Tetrazoline moiety were synthesized from phenobarbital. Phenobarbital 1 as raw starting material was reacted with acrylonitrile compound to give diacetonitrile derivative 2, this compound was treated in two ways, urea and thiourea to form barbituric acid derivatives containing oxadiazole and thiadiazole ring 3, 4 respectively. The Schiff bases derivatives 5, 6 (a-c) were synthesized from reacting the latter compounds with three aromatic aldehydes. In the final step, the barbituric acid derivatives containing 1,2,3,4Tetrazoline moiety 7, 8 (a-c) were prepared by cycloaddition reaction between different Schiff bases derivatives and sodium azide. The compounds were characterized by Melting point, 13 C-NMR, 1 H-NMR and FTIR techniques. Also, the result compounds were tested against two kinds of bacteria and two kinds of fungi. Most of the prepared derivatives were showed a high and clear effect against different types of bacteria and fungi. Molecular docking of final barbituric acid derivatives 7, 8 (a, b) were investigated with Molegro Virtual Docker (MVD).