Because of the illnesses and infections that pathogenic microorganisms can cause in their hosts, these microbes have become a major medical risk. As a result, researchers are looking for efficient antibiotics to cure these illnesses. Azo-naphthol dyes are often vividly coloured substances with a variety of industrial applications. Lately, they have demonstrated efficacy against specific strains of pathogenic microbes. The azo dye, 1-(3-nitrophenylazo)-2-naphthol, was synthesized in the current investigation through diazotization and azo coupling reactions between 3-nitroaniline and 2-naphthol. The NMR, IR, and UV-vis spectra data verified the presence of the molecular frameworks of the azo compound. The raw and 200 mg/mL concentrations of the sample inhibited the bacteria strains (Staphylococcus aureus and Escherichia coli) at the range of 0.2 mm to 0.8 mm while all other concentrations of the compound employed were resisted by the Aspergillus sp. However, the control antibiotics (Augmentin and Oflocitoxin) used, had little inhibitory effects on the fungus but were effective on the bacterial strains. The minimum inhibitory concentration was estimated at around 200 mg/mL. As no research has been carried out on the compound and also in this area, this compound can act as an antibacterial agent for various uses.