The purpose of this study was to perform a quantitative and qualitative evaluation of the biofilms formed on framework alloys in vitro. The biofilms formed by unfiltered fresh human saliva or Streptococcus mutans and/or Candida albicans on commercially pure titanium and gold-copper-platinum demonstrated higher amounts than other alloy and resin samples. In contrast, the silverpalladium-copper-gold showed a significantly decreased level of biofilm formation. Although the adhesion level of Streptococcus mutans on cobalt-chromium was high, that of Candida albicans was less extensive. A T-RFLP analysis and qualitative PCR of the microbes in the biofilms were performed. In a cluster analysis of all T-RFLP profiles, the cobalt-chromium pattern was integrated into one cluster. On qualitative PCR, the existence of microorganisms related to caries, preriodontitis and aspiration pneumonia was observed. Our results showed that the biofilm formation on each framework alloy was different in terms of both the quantity and quality.