The development of microalgal biofilms has received very limited study despite its relevance in the design of photobioreactors where film growth may be advantageous for biomass separation or disadvantageous in fouling surfaces. Here, the effects of species selection, species control, and substrate properties on biofilms of Scenedesmus obliquus and Chlorella vulgaris were investigated. Experiments were conducted in batch culture and in continuous culture modes in a flow cell. Cell growth was monitored using confocal laser scanning microscopy and gravimetrically. Species selection and species control had significant effects on biofilm development. On non-sterile wastewater, C. vulgaris shifted from primarily planktonic (23.7% attachment) to primarily sessile (79.8% attachment) growth. The biofilms that developed in non-sterile conditions were thicker (52 +/- 19 mu m) than those grown in sterile conditions (7 +/- 6 mu m). By contrast, S. obliquus attained similar thicknesses (54 +/- 31 and 53 +/- 38 mu m) in both sterile and non-sterile conditions. Neither species was able to dominate a non-sterile biofilm. The effect of substrate surface properties was minimal. Both species grew films of similar thickness (similar to 30 mu m for S. obliquus, < 10 mu m for C. vulgaris) on materials ranging from hydrophilic (glass) to hydrophobic (polytetrafluoroethylene). Surface roughness created by micropatterning the surface with 10 mu m grooves did not translate into long-term increases in biofilm thickness. The results indicate that species selection and control are more important than surface properties in the development of microalgal biofilms.