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Previous studies have reported that sanguinarine possesses inhibitory activities against several microorganisms, but its effects on mono- and dual-species biofilms of <i>C. albicans</i> and <i>S. aureus</i> have not been fully elucidated. In this study, we aimed to evaluate the efficacy of sanguinarine for mono- and dual-species biofilms and explore its ability to induce the hypha-to-yeast transition of <i>C. albicans</i>. The results showed that the minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC90) of sanguinarine against <i>C. albicans</i> and <i>S. aureus</i> mono-species biofilms was 4, and 2 &#956;g/mL, respectively, while the MIC and MBIC90 of sanguinarine against dual-species biofilms was 8, and 4 &#956;g/mL, respectively. The decrease in the levels of matrix component and tolerance to antibiotics of sanguinarine-treated mono- and dual-species biofilms was revealed by confocal laser scanning microscopy combined with fluorescent dyes, and the gatifloxacin diffusion assay, respectively. Meanwhile, sanguinarine at 128 and 256 &#956;g/mL could efficiently eradicate the preformed 24-h biofilms by mono- and dual-species, respectively. Moreover, sanguinarine at 8 &#956;g/mL could result in the transition of <i>C. albicans</i> from the mature hypha form to the unicellular yeast form. Hence, this study provides useful information for the development of new agents to combat mono- and dual-species biofilm-associated infections, caused by <i>C. albicans</i> and <i>S. aureus</i>.