Elevated atmospheric CO₂ increases plant C fixation, and much of the soluble C content of deciduous leaf litter entering streams is leached as dissolved organic C (DOC). The effects of DOC from trembling aspen (Populus tremuloides Michaux) leaf litter grown under elevated (ELEV = 720 ppm) and ambient (AMB = 360 ppm) CO₂ on stream periphyton were measured during a 35-d experiment in outdoor artificial stream chambers. Crayfish feeding preferences for periphyton grown in AMB and ELEV treatments were evaluated in short-term foraging trials using a Y-maze. Periphyton was sampled through time for ash-free dry mass (AFDM), chlorophyll a, total C:N, algal biovolume and species composition, and bacterial productivity and biomass. Leaf litter from plants grown under ELEV CO₂ produced higher concentrations of refractory DOC than did leaf litter from plants grown under AMB CO₂, and chlorophyll a concentrations were lower in periphyton enriched with ELEV DOC than in periphyton enriched with AMB DOC. ELEV DOC did not significantly affect bacterial productivity and biomass or total periphyton C:N, but cyanobacterial biovolume was higher in ELEV algal assemblages than in AMB algal assemblages after 35 d. AMB algal assemblages were dominated by the diatom Epithemia adnata var. proboscidea, which contains N-fixing endosymbionts. Orconectes virilis crayfish preferred AMB periphyton stimulus when offered the choice of AMB and ELEV stimuli or AMB and control stimuli. Our results suggest that DOC from trembling aspen leaf litter produced under ELEV CO₂ alters algal accrual and species assemblages of stream periphyton, and this shift in basal resource quantity and quality could affect feeding preferences of crayfish.