This study assessed the effect of zerumbone (ZER) against fluconazole-resistant (CaR) and -susceptibleCandida albicans(CaS) biofilms and verified the influence of ZER on extracellular matrix components. Initially, to determine the treatment conditions, the minimum inhibitory concentration (MIC), the minimum fungicidal concentration (MFC) and the survival curve were evaluated. Biofilms were formed for 48 h and exposed to ZER at concentrations of 128 and 256 µg/mL for 5, 10 and 20 min (n= 12). One group of biofilms did not receive the treatment in order to monitor the effects. The biofilms were evaluated to determine the microbial population (CFU/mL), and the extracellular matrix components (water-soluble polysaccharides (WSP), alkali-soluble polysaccharides (ASPs), proteins and extracellular DNA (eDNA), as well as the biomass (total and insoluble) were quantified. The MIC value of ZER for CaS was 256 μg/mL, and for CaR, it was 64 μg/mL. The survival curve and the MFC value coincided for CaS (256 μg/mL) and CaR (128 μg/mL). ZER reduced the cellular viability by 38.51% for CaS and by 36.99% for CaR. ZER at 256 µg/mL also reduced the total biomass (57%), insoluble biomass (45%), WSP (65%), proteins (18%) and eDNA (78%) of CaS biofilms. In addition, a reduction in insoluble biomass (13%), proteins (18%), WSP (65%), ASP (10%) and eDNA (23%) was also observed in the CaR biofilms. ZER was effective against fluconazole-resistant and -susceptibleC. albicansbiofilms and disturbed the extracellular matrix.