Introduction. Candida albicans can produce a complex, dynamic and resistant biofilm on the surface of dental materials, especially denture base acrylic resins and temporary soft liners. This biofilm is the main aetiological factor for denture stomatitis, an oral inflammatory condition characterized by chronic and diffuse erythema and oedema of the denture bearing mucosa. Gap Statement. There is no consensus in the literature regarding the best method to detach biofilms from dental materials. In order to assess the antifungal efficacy of new materials and treatments, the biofilm needs to be properly detached and quantified. Aim. This study compared different methods of detaching C. albicans biofilm from denture base acrylic resin (Vipi Cril) and temporary soft liner (Softone) specimens. Methodology. Specimens of each material were immersed in an inoculum of C. albicans SC5314 and remained for 90 min in orbital agitation at 75 r.p.m. and 37 °C. After the removal of non-adherent cells, the specimens were immersed in RPMI-1640 medium for 48 h. Biofilm formation was evaluated with confocal laser scanning microscopy (n=5). Then, other specimens (n=7) were fabricated, contaminated and immersed in 3 ml of sterile phosphate-buffered saline (PBS) and vortexed or sonicated for 1, 2, 5, or 10 min to detach the biofilm. The quantification of detached biofilm was performed by colony-forming unit (c.f.u.) ml⁻¹ count. Results were submitted to one-way analysis of variance (ANOVA)/Tukey HSD test (α=0.05). Results. A mature and viable biofilm was observed on the surfaces of both materials. For both materials, there was no significant difference (P>0.05) among detachment methods. Conclusion. Any of the tested methods could be used to detach C. albicans biofilm from hard and soft acrylic materials.