Microbial surface active compounds (SACs) were isolated from various environmental sources in Thailand. Isolates were screened for SACs production in different carbon sources (crude glycerol, commercial sugar, decanter, glucose, molasses, used palm oil, and used lubricating oil) by using drop-collapsing test and emulsification activity. Molasses produced the highest number of positive results (23 of 25 isolates). Twenty-one isolate strains produced emulsions with xylene, and 15 exhibited high emulsion-stabilizing capacity, maintaining more than 50 % of the original emulsion volume for 24 h, and six isolate strains reduced the growth medium surface tension to 40 mN/m. The phylogenetic position of these 25 isolates was evaluated by 16S rRNA gene sequence analysis. The production of microbial SACs was determined for strains representative of 16 different bacterial genera, in which ten genera (Blastococcus, Erysipelothrix, Humicoccus, Methylophilus, Microlunatus, Nevskia, Pectinatus, Rubrimonas, Selenomonas, and Stenotrophomonas) were firstly reported as SAC-producing strains. Overall, the new SAC-producing strains isolated in this study display promising features for the future development and use in economically efficient industrial-scale biotechnological processes.