The metabolites produced by Bacillus spp. have a strong ability to inhibit pathogenic fungi. Because of the similar structure and molecular weight of the metabolites, it is necessary to purify and identify the metabolites by high-precision separation strategy. There are many methods for the purification and structure confirmation of Bacillus metabolites, e.g., high-performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan), reverse-phase chromatography (RPC, Shimadzu, Kyoto, Japan), gel permeation chromatography (GPC, Shimadzu, Kyoto, Japan), mass spectrometry (MS, Bruker Daltonics, Bremen, Germany), nuclear magnetic resonance (NMR, Bruker, Madison, WI, USA) and infrared (IR) spectroscopy (Jasco Analytical, Tokyo, Japan) etc. The marine bacteria Bacillus subterraneus 11593 can produce a new indole alkaloid. By detailed analysis of its NMR spectroscopic data, and further by the theoretical ECD (electronic circular dichroism) calculations, the absolute configuration of the new compound was determined [30]. The metabolites of Burkholderia sp.by IR spectroscopy (Shimadzu, Japan) were analyzed. The stretching vibration of glycosidic bonds in the fingerprint area (1200 cm−1 to 800 cm−1) of the IR spectroscopy confirmed that the biosurfactant produced by endophytic bacteria is glycolipid [31]. For some metabolites, simple extraction methods can also achieve better results. Marine bacterium B. subtilis has an antibacterial membrane effect against C. albicans. The metabolite was 5-hydroxymethyl-2-furaldehyde (5HM2F) by ethyl acetate extraction and mass spectrometry analysis [32]. A new bioactive compound from Bacillus megaterium was extracted, purified and identified. The bioactive compounds were extracted with n-butanol and purified on a TLC plate. Further structural confirmation indicated that it is a cyclic polypeptide with similar structure to bacitracin and broad-spectrum antibacterial activity [33]. The anti-C. albicans bacteriocin produced by Bacillus Sh10 is purified by precipitation and gel chromatography. Through the purification process, the specific activity increased by 3.68 times, and the total activity recovery rate was 20.66%. The molecular weight of the compound was determined by SDS-PAGE, and the bacteriocin was analyzed by LC/MS/MS (Agilent Technologies, Santa Clara, CA, USA) [26].