New findings on the application of microbial extracellular polymeric substances (EPS) in minimizing harbor siltation have been reported recently. However, the key components of EPS influencing fluid mud anti-settlement properties have not been well elucidated. The relationships between EPS contents and anti-settleability of mud were thereby investigated. Results showed that, after cultivation with nutrients, the settling velocity of mud increased with the increase of crude EPS contents, indicating anti-settleability of mud was positively correlated to EPS content. The yield and composition of EPS were analyzed by extraction with five different methods. It was shown that polysaccharide and protein were the main components of EPS with verification by Fourier transformed infrared spectroscopy, and polysaccharide was the dominant composition in bound EPS. Thus, it was speculated that polysaccharide in EPS was the key component influencing the anti-settlement properties of mud. Moreover, ultrasound process was the optimum protocol for extracting EPS from mud.