This study aims to elucidate the mechanisms governing the harvesting efficiency of Chlorella vulgaris by flocculation using a cationic polymer. Flocculation efficiency increased as microalgae culture matured (i.e. 35–45, 75, and > 97% efficiency at early, late exponential, and stationary phase, respectively. Unlike the negative impact of phosphate on flocculation in traditional wastewater treatment; here, phosphorous residue did not influence the flocculation efficiency of C. vulgaris. The observed dependency of flocculation efficiency on growth phase was driven by changes in microalgal cell properties. Microalgal extracellular polymeric substances (EPS) in both bound and free forms at stationary phase were two and three times higher than those at late and early exponential phase, respectively. Microalgae cells also became more negatively charged as they matured. Negatively charged and high EPS content together with the addition of high molecular weight and positively charged polymer could facilitate effective flocculation via charge neutralisation and bridging.