Based on demulsification performance, twenty biodemulsifier-producing strains were isolated from various environmental sources. Five of them achieved nearly or over 90% of emulsion breaking ratio within 24 h. With the aid of biochemical and physiological tests and 16S rDNA analysis, these isolates were classified into eleven genera, in which six genera (Brevibacillus sp., Dietzia sp., Ochrobactrum sp., Pusillimonas sp., Sphingopyxis sp. and Achromobacter sp.) were firstly reported as demulsifying strains. Moreover, with data in this study and other literatures, a phylogenic tree was constructed, showing a rich diversity of demulsifying bacteria. Half of these bacteria belong to Actinobacterale order, which is famous for hydrocarbon degradation and biosurfactant biosynthesis. However, some strains in the same genera differed remarkably in demulsifying capability, surface properties and biochemical and physiological characteristics. This implied the biosynthesis and composition of biodemulsifier were more complicated than expected.