Nano-boosted thymoquinone: moving beyond antibiotics to inhibit <i>Staphylococcus aureus</i> biofilms

<i>Staphylococcus aureus</i> is a high-priority pathogen requiring novel antimicrobial strategies. Thymoquinone (TQ), a bioactive compound from <i>Nigella sativa</i>, exhibits antimicrobial and antibiofilm properties, but its precise mechanisms remain unclear. This study evaluated free and nano-encapsulated TQ against multidrug-resistant <i>S. aureus</i> isolates. Nano-encapsulation enhanced biofilm penetration, with TQ significantly reducing extracellular DNA (eDNA) at sub-MIC levels without affecting initial adhesion, exopolysaccharides, or enzymatic virulence. Comparative analysis with vancomycin, ciprofloxacin, and azithromycin confirmed TQ’s potent antibiofilm activity. Notably, TQ downregulated <i>crtN</i>, essential for staphyloxanthin biosynthesis, and <i>msaB</i>, a key biofilm and stress response regulator. However, at MIC and super-MIC levels, TQ paradoxically increased staphyloxanthin, suggesting a concentration-dependent oxidative stress response. We propose that TQ disrupts biofilm integrity by modulating the <i>msaABCR</i> operon. These findings highlight nano-TQ’s therapeutic potential for biofilm-associated infections and underscore the role of natural compounds in combating multidrug-resistant pathogens. Graphical abstract illustrating the hypotheses of the study, experimental workflow, and key conclusions aimed at evaluating the antibiofilm activity of thymoquinone (TQ) against <i>Staphylococcus aureus</i>. Graphical abstract was generated using BioRender.