Eliciting oxidative stress and interference with biofilm formation by silver nanoparticles (AgNPs) comprise some of the postulated mechanisms of bacteria destruction. Plant extracts are commonly employed both as reducing agents and stabilizers of green-synthesized AgNPs. This yields a broad nanoparticle size distribution and unpredictable coating arising from diverse biomolecules and oxidation products in the final reaction mixture, which makes the comparison of different experiments exploiting green synthesised AgNPs as antimicrobial agents tricky. The current study synthesised citrate-capped silver nanoparticles (CtAgNPs) with a diameter of 37.9±8.6 nm functionalized via ligand exchange with betanin. Subsequently, the betanin silver nanoparticles (BtnAgNPs) ability to perturb the cellular oxidative stress defence mechanism of Staphylococcus aureus American Type Culture Collection (ATCC) 12600 and biofilm-forming properties were evaluated. Treatment with a minimum inhibitory concentration (MIC) of BtnAgNPs (12.5 ppm) promoted reactive oxygen species accumulation and significantly reduced superoxide dismutase (SOD) and catalase activities. Furthermore, BtnAgNPs at sub-minimum inhibitory concentrations (SubMICs) to MIC range (0.39-12.5 ppm) inhibited the establishment of biofilms in a concentration-dependent manner. The study advocates for synthesis protocols that produce precise-sized nanoparticles followed by selective biomolecule functionalization for better antimicrobial efficacy comparative studies between AgNPs bearing different coatings in future experiments.