Microbiological flocculation in wastewater by a strain of Bacillus mucilaginosus was examined and the flocculation mechanism was investigated using Coomassie brilliant blue reaction, Anthrone reaction, thin-layer chromatography (TLC), scanning electronic microscope, and infrared spectroscopy. The GY03 strain, isolated from farmland soil, was identified as B. mucilaginosus by its morphology and 16S rDNA sequence. Cultivated in a nitrogen-free medium, the flocculation material (bacterium-mineral complex) was used to treat domestic, brewage, and pharmaceutical wastewater. On the basis of one-way experimental results, orthogonal experiments were carried out and the optimum processing conditions of the microbial flocculants were obtained. In the optimal conditions, the maximal removal ratio of COD in domestic, brewage, and pharmaceutical wastewater reached 74.6%, 70.5%, and 66.2%, respectively; the maximal removal ratio of BOD was 42.3%, 77.4%, and 41.7%, respectively; and the maximal removal of SS amounted 93.3%, 93.6%, and 88.4%, respectively. The treatment to kaolin suspended liquid by the GY03 strain may act as a model of studying microbial flocculation mechanisms in which bridging and charge neutralization hypothesis were proposed as the critical reasons based upon the experimental observations.