A bacteria strain Citrobacter W4 isolated from the microalgae sewage culture system showed flocculating activity against Chlorella pyrenoidosa. In this work, operation parameters under outdoor conditions were optimized. When the bacterial-algal ratio was 4:1, G value was 26.30 s−1, and harvesting time was 6 h, the harvesting efficiency achieved 87.37 ± 2.96% without ions addition and pH adjustment. The microbial community structure analysis showed Citrobacter W4 was dominant in the harvesting process. Flocculating active substances were on the surface and metabolites of Citrobacter W4. The main component of bacteria flocculating active substances was protein. Polysaccharides and carboxylic acid also promoted flocculation. The flocculation mechanisms were mainly adsorption bridging, net catching, and sweeping, not electric neutralization. The quality of FAMEs was improved after flocculation. The cost of 1 kg dried microalgae flocculated by Citrobacter W4 was $1.35. The novel flocculating bacteria showed the potential to harvest microalgae cost-effectively and environmentally friendly. Display omitted •The harvesting efficiency achieved 87.37% flocculated by Citrobacter W4.•Protein was the main component of bacteria flocculating active substances.•Citrobacter W4 flocculated by adsorption bridging, net catching and sweeping.