Dehydroeffusol (DHE) is a natural phenanthrene compound derived from Juncus effusus L., traditionally used for its antimicrobial properties. Despite its historical use, its antibacterial efficacy and underlying mechanisms have not been fully explored.

To evaluate the antibacterial activity, safety profile, and mechanism of action of DHE against Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), and to assess its potential as a therapeutic agent.

DHE was isolated and its antibacterial effects were assessed using broth microdilution, time-kill assays, and biofilm inhibition assays. The cytotoxicity and hemolytic activity of DHE were evaluated in vitro using several cell lines and red blood cells. Mechanistic studies included scanning electron microscopy, membrane potential and permeability assays, network pharmacology analysis, molecular docking, and untargeted metabolomic profiling.

DHE exhibited appreciable bactericidal activity against S. aureus and MRSA with minimal cytotoxicity. It inhibited biofilm formation, disrupted bacterial membranes, and maintained bactericidal activity without rapid resistance, while synergizing with ciprofloxacin, streptomycin, and azithromycin. Network pharmacology and docking identified 56 infection-related targets (hub proteins EGFR, BCL2, HSP90AA1/B1, ESR1, SRC) enriched in PI3K–Akt/ErbB/EGFR pathways, supporting additional modulation of host infection-related signaling. In MRSA, untargeted metabolomics showed broad disturbances in amino acid, energy, and nucleotide metabolism, indicating disruption of essential biosynthetic and energy pathways. In vivo, DHE significantly reduced bacterial load and alleviated tissue damage in a murine MRSA infection model, demonstrating measurable protective effects.

DHE is a promising natural antimicrobial agent with notable activity against S. aureus and MRSA. Its mechanism of action involves membrane disruption and biofilm inhibition, along with preliminary safety in vitro, supporting its potential for further development as an effective antimicrobial agent.