Two TaqMan® qPCR assays were developed to specifically quantify the absolute abundance of Scenedesmus obliquus and Chlorella vulgaris in mixed-species algal biofilms by targeting the psbA gene. Standard curves were developed with amplification efficiencies of 92.4% and 96.6% for S. obliquus and C. vulgaris, respectively, and an R2 value of 0.99 for both. Calibration curves for estimating absolute cell abundances resulted in slopes of 0.98 and 1.11 for C. vulgaris and S. obliquus, respectively, and an R2 value of 0.95 for both. The assays were applied to cultivated mixed-species biofilms and approximately 107 cells of each algal species were quantified when 107 cells were added into biofilms. The developed qPCR assays were concluded to be specific and accurate for the quantification of S. obliquus and C. vulgaris in mixed-species biofilms. This will contribute to the control and optimization of algal cultivation systems for the production of algal biofuels and bioproducts.