Purpose: This study evaluates the antibiofilm action of 2.5 mg/mL peracetic acid (PA), 0.5 mg/mL cetylpyridinium chloride (CPC), and 160 mg/mL N-Acetylcysteine (NAC) against multispecies biofilm of Streptococcus mutans, Staphylococcus aureus, Candida albicans, and Candida glabrata, developed on surfaces of heat-polymerizing acrylic resin (AR) and cobaltchromium (Co-Cr) alloy. Materials and Methods: A multispecies biofilm was grown on the surface of AR and Co-Cr specimens (Ø 12×3mm). After biofilm maturation, the specimens were immersed in experimental solutions and evaluated through biofilm viability (CFU) (n=9), biofilm metabolic activity (XTT) (n=9), biofilm-covered areas (Live/Dead) (n=2), effects on the extracellular polymeric substance (EPS) (n=2) and biofilm morphology (n=1). Data were analyzed by ANOVA and the Tukey post-test or Kruskal-Wallis followed by the Dunn post-test (α=.05). Results: Overall, all evaluated solutions impacted biofilm viability. PA presented wider activity by reducing CFU of all microorganisms on both surfaces, XTT (P<.001) and Live/Dead (P<.001). NAC had a notorious effect in reducing the viability of bacteria without affecting the yeasts. NAC reduced XTT on AR (P=.006) and Co-Cr (P=.003) but did not reduce the aggregated biofilm layer. CPC had distinct effect according to the surface, being most effective in reducing CFU on AR than the Co-Cr surface. However, it did not influence XTT, and the amount of residual aggregated biofilm. Conclusions: PA provided the greatest antibiofilm action, while CPC and NAC showed intermediate action. Nonetheless, no solution was able to completely remove the biofilm adhered to the surfaces of heat-polymerizing AR and Co-Cr alloy.