Polyaniline-co-Phenylenediamine Nanocomposite has been prepared in the aqueous medium using sodium dodecyl benzene sulfonate (DBSNa) and hydroxypropyl cellulose (HPC) as a surfactant. The tests used in this research to characterize the products were SEM, TEM, FTIR, UV-visible and TGA for morphology, particle size, chemical structure and stability. The results confirm that the spherical nanocomposites (40–90 nm) were formed with high thermal stability. It is shown in the results that the physicochemical properties of poly(alkyl substituted anilines) are depended on the substituent groups that are bonded to N-position. The prepared nanocomposites were then tested for the antibacterial properties against three pathogenic strains. The antibacterial properties of nanocomposites were investigated by disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentrations (MBC), and bactericidal kinetic methods. The disk diffusion result indicated that the diameter of the inhibition zones of PAn/PDA-HPC nanocomposite was 9, 11 and 10 mm against E. coli, P. aeruginosa, and S. aureus respectively. It was found that the value of MIC of PAn/PDA-HPC nanocomposite against E. coli, P. aeruginosa and S. aureus were 2.5, 1.25 and 2.5 mg/ml respectively. The evaluation results revealed the PAn/PDA-HPC nanocomposite exhibited excellent inhibitory activity against both Gram-negative and gram-positive bacteria.