The new azo dye (A1), that named (2S,5R,6R)-6-((R)-2-((3-carboxy-4-hydroxyphenyl)diazenyl)-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid was prepared. And then characterized using m.p., IR, UV-visible and elemental analysis CHN. Analytical studies of (A1) were showed that the best solubility was in methanol, without straying from the linear connection, owing to suggest The major factor controlling the shift in absorption beaks is the influence of the dielectric constant. However, the pH effect of (A1) in a range of buffer solution was revealed that the appropriate pH values were discovered to be in the pH12 range, and that three isopiestic points were achieved We determined the ionization (pKa) and protonation (pKp) constants. due to designate the ionization strategy in acidic and basic media. The synthetic azo dye remained harmless, nontoxic and do not has any heamolysis effect in the human blood cells. Therefore, the biological activity of (A1) against different bacteria, (Staphylococcus Aureus, Escherichia Coli, Bacillus Cereus and Pseudomonas Aeruginosa) and two different fungi (Aspergillus Albicans and Candida Albicans) were studied. The results were indicated that the dye can affect the fungi more than bacteria. Owing to recommend the new azo dye as novel drug or chemical sanitizer for these microorganisms.