Lawsone, (2-hydroxy-1,4-naphthoquinone or HNQ), is known for its antimicrobial activity, corrosion inhibition and metal chelation properties. We describe here a simple methodology to functionalize Fe₃O₄ nanoparticles with lawsone to produce Fe₃O₄-Cysteine-HNQ nanocomplex, using cysteine as a linker. The characterization of the nanocomplex is done using Fourier Transform Infrared (FTIR) spectroscopy, Thermogravimetric Analysis (TGA), Dynamic Light Scattering size measurement, zeta potential and contact angle measurement. The zeta potential of uncoated nanoparticles and Fe₃O₄-Cysteine-HNQ nanocomplex is found to be +48 mV and −38 mV respectively. The reversal of surface charge from positive to negative corroborates the presence of lawsone monolayer at particle interface. Further, TGA result shows a weight loss of 32% for these nanocomplexes due to the decomposition of lawsone and cysteine present on the nanoparticles. The FTIR spectrum of the nanocomplex exhibits CC and CO stretching bands of lawsone, with a slight shift in their position, which confirms the interaction of lawsone with the cysteine coated nanoparticles. Fe₃O₄-Cysteine-HNQ nanocomplexes exhibit interesting properties such as increased wettability (contact angle ∼20°) on stainless steel substrate and antimicrobial activity against Staphylococcus aureus, with a minimum inhibitory concentration of 700 μg mL⁻¹. These results show that lawsone functionalized magnetite nanoparticles can be used as response stimulus antimicrobial agents.