Purpose Dental prosthetic and orthodontic appliances are transported from the clinic to the laboratory for additions and repairs. These appliances, containing microbes from the oral flora, are a high risk for cross‐contamination. The aim of this study was to evaluate the effect of chemical and ultrasound disinfection against two in vitro biofilms and an in vivo formed biofilm grown on unprepared and polished polymethyl methacrylate (PMMA) surfaces. Materials and Methods Rough and polished self‐curing PMMA surfaces were infected with strains of both Candida albicans and Streptococcus oralis. After incubation, the samples were treated with different disinfection methods, including ultrasound treatment for both 15 and 30 seconds, and immersion in glutaraldehyde and alcohol‐based chemical disinfectants (MD520 and Minuten, respectively). The disinfecting efficacy was assessed by colony forming units (CFU) analysis and by scanning electron microscopy (SEM). Furthermore the adequacy of bacterial elimination of application of 30‐second ultrasound and MD520 was assessed on PMMA retrieved from ten volunteers by CFU analyses. ANOVA with p = 0.05 followed by the Tukey post hoc test and the Student t‐test was used to analyze the data. Results The ultrasound treatment for 30 seconds, MD520, and Minuten were the most effective disinfectant methods as they reduced the microbial counts compared to the control (p < 0.05) as shown in the in vitro analyses. S. oralis adhered more to rough acrylic resin surfaces (p < 0.05). Ultrasound treatment was the most effective way to reduce microbial counts on PMMA exposed to oral flora (p = 0.043). Conclusion Ultrasound treatment for 30 seconds was effective against C. albicans, S. oralis, and the oral flora as shown by testing microbial growth on agar plates and SEM.