Staphylococcus aureus remains a major human pathogen and biofilm formation and antibiotic resistance make the treatment more difficult. Helianthus tuberosus L. (Jerusalem artichoke) is a phenolic-rich plant with reported antimicrobial properties, but its antibiofilm activity and putative AgrA-targeting constituents are poorly documented. In this study, an ethanolic H. tuberosus extract (SS-WU-1) was investigated against S. aureus ATCC 25923 and methicillin-resistant S. aureus (MRSA) ATCC 43300 using an integrated in vitro–in silico workflow. Minimum inhibitory concentration (MIC) values were >2.250 mg/mL and 1.125 mg/mL, respectively, and Minimum Bactericidal Concentration (MBC) values were >2.250 mg/mL for both strains. Sub-inhibitory treatment significantly reduced MRSA biofilm formation with 96.6 ± 0.5% inhibition at 1/2× MIC. Scanning electron microscopy showed decreased bacterial coverage, greater dispersal of cells and altered biofilm architecture. UHPLC-ESI-QTOF-MS/MS profiling yielded 677 tentatively annotated molecular features (481 ESI+ and 196 ESI−). A multiple linear regression QSAR model trained on 15 experimentally characterized AgrA inhibitors showed good fit (R² = 0.8749; Q²_LOOCV = 0.7017). Virtual screening and applicability-domain assessment prioritized P273, P385, and P348 for molecular docking against the AgrA LytTR domain. These compounds represent computationally prioritized candidates for experimental validation. Inhibition of biofilm formation without concomitant growth inhibition suggests a potential anti-virulence effect relevant to the prevention of device-associated infection. SS-WU-1 therefore represents a promising source for further investigation of antibiofilm phytochemicals, although the potential involvement of AgrA remains a hypothesis requiring experimental validation.