The aim of the study is to develop advanced antibacterial agents as nanoparticles instead of antibiotics due to the emergence of antimicrobial resistance. Pseudomonas aeruginosa is capable of causing many diseases, including serious bacterial pneumonia. There is a need for an efficient antibacterial agent to kill these pathogens. The objective of the study is the synthesis of advanced antibacterial agents as nanoparticles for biomedical applications that can play a vital role to kill Gram-negative bacteria (Pseudomonas aeruginosa). A novel fabrication growth of hydrophilic spiky gold nanoparticles (SGNPs) via reduction method is reported. Surface plasmon resonance peak of the synthesized SGNPs was tuned under near infrared range. The SGNPs have anisotropic and spiky morphology with 68 nm size and -58 mV surface charge and are pure, having adsorption of the organic material. Pseudomonas aeruginosa treated with synthesized SGNPs showed 60% bacterial death at the concentration of 100 μM. This work consists of novel synthesis of SGNPs via safe and simple reduction method. The synthesized SGNPs exhibit strong antibacterial activity against the Gram negative bacteria Pseudomonas aeruginosa measured using microplate assay test. The result showed that these SGNPs are ideal for biomedical applications. Copyright© Bentham Science Publishers; For any queries, please email at [email protected].