(1) Background: Rapidly increasing antibiotic resistance is one of the greatest threats to global health, affecting individuals regardless of age. Medicinal plants are widely used in traditional medicine to prevent and attenuate infectious conditions with minimal adverse effects. However, only a few have been phytochemically investigated for their medicinal properties and subsequent biological activities. Syncarpia hillii, a plant traditionally used by Indigenous Australians to treat sores, wounds, and skin infections, is no exception. (2) Methods: Primary extracts obtained from mature S. hillii leaves were evaluated for their antibacterial potential against 19 bacterial strains. The methanol extract was subjected to compound isolation and identification due to its preliminary bactericidal efficacy. (3) Results: Staphylococcal species were the most susceptible bacterial strain with a MIC value of 0.63 mg/mL to the S. hillii methanol extract. Quercetin-3-O-β-D-glucuronide and shikimic acid isolated from S. hillii methanol leaf extracts exhibited enhanced antibacterial effects against the tested bacteria with quercetin-3-O-β-D-glucuronide eliciting a MIC value of 0.78 µg/mL against E. faecalis. (4) Conclusions: S. hillii leaves are comprised of bioactive compounds that are bactericidal against several Gram-positive and Gram-negative bacteria.

A virtual screening approach based upon a combination of docking and pharmacophore methods was utilized on a library of 1.4 million molecules to identify novel antimicrobial agents, which may potentially act via inhibition of the caseinolytic protease. Using this method, compound 6 was found to be bactericidal against three staphylococcal species (minimum inhibitory concentration (MIC)=4–16 μg/mL). Further, subsequent structural optimization of 6 led to the identification of compound 24, which was shown to be the most potent analog within the series (MIC=4 μg/mL) and outperforming the antibiotic controls for two of the staphylococcal species. The newly discovered antimicrobial agent (24) demonstrated excellent in silico ADME properties and was non‐toxic when tested on two human skin cell lines. As such, compound 24 has the potential for use as a lead molecule in the development of a novel class of antimicrobial agents.