ABSTRACT Biofilm formation is a key survival strategy among microorganisms and a major factor contributing to chronic and treatment‐resistant infections. Staphylococcus aureus is a key biofilm‐forming pathogen associated with persistent and life‐threatening infections. As part of our ongoing search for novel anti‐biofilm agents from Basidiomycota of tropical rainforests, we investigated the metabolome of the African fungus Neonothopanus nambi . This study led to the isolation of four dimeric aristolane‐type sesquiterpenoids, aurisins D, B, A, and G ( 1 – 4 ), along with two monomeric sesquiterpenoids, nambinone C ( 5 ) and axinysone B ( 6 ), and methyl 4‐butyramidobenzoate ( 7 ). All compounds were assessed for their antimicrobial and cytotoxic activities, as well as their ability to inhibit and eradicate S. aureus biofilms. The dimeric sesquiterpenoids ( 1 – 4 ) exhibited potent antibiofilm effects, with aurisin B ( 2 ) inhibiting biofilm formation by over 70% at a concentration of 1 µg/mL, well below its minimum inhibitory concentration. Confocal laser scanning microscopy further confirmed the pronounced anti‐biofilm effects of aurisins D and B ( 1 and 2 ). Structure–activity relationship analysis suggests that both dimerization and hydroxylation contribute to enhanced activity. Despite some cytotoxic effects, these dimeric aristolane‐type sesquiterpenoids from N. nambi represent promising leads for the development of novel anti‐infective strategies targeting S. aureus biofilms, with potential applications beyond systemic use.