Periphyton cultivation systems have few controls over species composition, thereby limiting their applications for production of biomass. A promising approach for controlling species composition in cultivation systems is through design of substratum characteristics. 3-D printing is used to design substratum topographic sections to test for selectivity of colonization of periphyton algae in streams. Experimental tiles were designed with six different topographic sections composed of tightly-packed hemispheres over a range of diameters from 100 to 2000 μm. These tiles were deployed in a local stream for 30 days, retrieved and analyzed for species occurrence and relative abundance on each of the tile sections. Twelve species of periphyton algae in four divisions were identified across all topographic sections. The distribution and relative abundance of these species were found to vary as a function of topographic feature size, with the greatest diversity observed on the surfaces with topographic feature sizes of 500 μm. Of the twelve identified species, two exhibited abundance patterns that were significant. Microspora wileana displayed a preference for surfaces with topographic feature sizes < 500 μm, and Stigeoclonium tenue displayed a preference for surfaces with topographic feature sizes less than or equal to 100 μm and greater than or equal to 1500 μm. These results suggest that substratum design using 3D printing or other technologies may be useful to influence species composition and dominance relationships in mixed communities in engineered periphyton cultivation systems.