This study was conducted to examine the production of bioflocculants using agricultural wastewater as a fermentation feedstock under different temperatures and incubation times. The mechanism of flocculation was studied to gain a detailed understanding of the flocculation activity. The highest bioflocculant yield (2.03 g/L) at a temperature of 40 °C was produced in a palm oil mill effluent medium (BioF-POME). Bioflocculant produced from a fermented SME medium (BioF-SME) showed the highest activity. The flocculation tests for colour and turbidity removal from lake water indicated that BioF-SME and BioF-POME performed comparably to commercial alum. Analyses of the bioflocculants using liquid chromatography-mass spectrometry (LC-MS) found that the bioflocculants contained xylose and glucose. The mechanism study showed that flocculation occurred through charge neutralization and interparticle bridging between the bioflocculant polymer and the particles in the lake water. Thus, agricultural wastewater can be used as a fermentation feedstock for high-quality bioflocculants.