The use of aqueous two-phase systems (ATPSs) and each system's individual phase-forming species to prevent Streptococcus sanguis attachment onto hydroxyapatite discs was explored. The strategy that we followed was to attach the cells to a solid surface in the presence of an additional interface. Conditions under which, simultaneously, the phase-forming species form two phases and the cells proliferate were identified. Growth curves were constructed in the presence of various polymers and salts commonly used to prepare ATPSs. Several aqueous two-phase systems were selected such that bacterial growth was comparable to that observed in pure medium. Cells were allowed to attach to hydroxyapatite discs for 7 days in the presence of varying concentrations of media, media with polymer, media with salt, and media with ATPS. Streptococcus sanguis attachment to the disks was evaluated by scanning electron microscopy. The addition of a PEG/Na2SO4 ATPS to high concentrations of yeast-tryptone (YT) media (>65%) and of a PEG/MgSO4 ATPS to nutrient-limited media reduces surface coverage of S. sanguis to less than 10%. Comparison of the attachment levels for the systems containing PEG/Na2SO4 to media containing the individual phase-forming species and to the YT reference systems indicated that nutrient availability did not affect attachment.