The bioprospecting of potentially mixotrophic microalgae in a constructed wetland was conducted. A locally isolated microalga, Chlorella sp., was grown to determine the effect of temperature, aeration rate, and cultivation time on simultaneous biomass production and nutrient removal from piggery wastewater using central composite design (CCD). The most important variable for the biomass productivity of Chlorella sp. was aeration rate, while that for lipid content and nutrient removal efficiency was cultivation time. Total nitrogen (TN) and total phosphorus (TP) removal efficiencies were higher than that of chemical oxygen demand (COD) from piggery wastewater. The CCD results indicate that the highest biomass productivity (79.2 mg L−1 d−1) and simultaneous nutrient removal efficiency (TN 80.9%, TP 99.2%, COD 74.5%) were obtained with a cultivation temperature of 25 °C, a cultivation time of 5 days, and an air aeration rate of 1.6 L L−1 min−1. Palmitic acid (C16:0) and linoleic acid (C18:2) were both abundant in Chlorella sp. cells under mixotrophic cultivation with piggery wastewater.