Ecological wastewater treatment systems that incorporate aquatic plants (like duckweed) have the potential to recover nutrients and produce high-quality protein, simultaneously alleviating two global issues: hunger and lack of sanitation. Although protein production by duckweed in simple lagoon systems and laboratory trials has been reported, its physiology throughout more complex wastewater treatment systems containing a range of different environmental conditions has not been examined. In this study, a duckweed co-culture (Lemna japonica/minor and Wolffia columbiana) was grown on wastewater from four different stages of a pilot-scale ecological treatment system. Contrary to the literature, the protein content of duckweed did not consistently increase with increasing aqueous nitrogen concentrations, but rather appeared to also be dependent on chemical and microbial interactions. This study indicates that with proper management, duckweed grown in ecological wastewater systems can sustainably produce protein at rates exceeding those of common land-grown forage crops (10.1 ton ha−1 yr−1).