Herein, an effort was made to investigate the antibacterial potential of agarose nanoparticles (ANPs) and poly(quaternary ammonium) modified ANPs (mANPs) against Escherichia coli (gram-negative bacterium) and Staphylococcus aureus (gram positive bacterium) in liquid systems as well as on agar plates. ANPs were synthesized by nanoprecipitation technology and characterized by XRD, TEM, TGA, DTA and DLS. The particle size estimated was 30 nm while atomic force microscopy was used to observe the interaction of ligand on ANPs. Antimicrobial characterization was monitored by colony forming units (CFU) as a function of ANPs concentration on agar plates. It was observed that ANPs showed 15 × 109/ml CFU after 24 hours of incubation at 20 mM ANPs concentration while the modified ANPs exhibited 21 × 109/ml CFU under similar incubation conditions. Moreover, zone of inhibition (ZOI) was 2.9 and 3.8 cm, respectively for E. coli by ANPs at 0.2 and 0.4 mM, respectively while it was 3.2 and 3.8 cm respectively by modified ANPs under similar conditions. Similarly, ZOI for S. aureus by ANPs at 0.2 and 0.4 mM was observed at 3.1 and 4.0 cm, respectively, while these values were 3.5 and 4.1 cm, respectively for modified ANPs under similar incubation conditions.