Forty-eight out of sixty-eight bacterial strains isolated from traditional fermented soybean samples (Tuong Ban) showed moderate to high fibrinolytic enzyme activity. Bacillus sp. HY6 had the highest fibrinolytic enzyme activity of 80.4 ± 1.2 FU/mL. The strain was selected for improving fibrinolytic activity via UV irradiation and chemically induced mutagenesis using Ethidium Bromide (EtBr) and Ethyl Methane Sulfonate (EMS). After UV-irradiation, the HY6-derived mutant, designated as strain U5_90.5, exhibited the highest fibrinolytic enzyme activity of 127 ± 5 FU/mL, about 1.6 times higher than that of the wild type HY6 strain. Strain U5_90.5 after UV mutation was subsequently subject to chemically induced mutation by EtBr and EMS. This double mutation resulted in strain ES4 which has the highest fibrinolytic enzyme activity of 404 ± 4 FU/mL. The enzyme production capability of the strain was stable after 10 generations which renders the strain a potential fibrinolytic enzyme producing strain for commercial applications. This result indicated that the Bacillus sp. ES4 could be a potential commercialized fibrinolytic enzyme producing bacterium.