The objective of this research was to perform screening of biosurfactant-producing bacteria from diesel contaminating areas in and around Madurai and Sivagangai Districts. Eight sampling sites were chosen in total, four each from Sivagangai and its surroundings and Madurai and its surroundings. The isolates were cultured in nutrient broth and Bushnell Haas (BH) liquid medium with 1% (v/v) diesel, and their extracts were evaluated according to drop collapse, oil dispersion, emulsification, and blood agar tests. From two hundred and eighty four isolates, 17 isolates were selected, screened for biosurfactant production. The extracellular biosurfactant production pointed out the 3 most efficient bacteria that presented high production. The three isolates were identified by 16S rDNA gene sequencing, indicating the presence of two different genera, Enterobacter and Lysinibacillus. Cluster analysis classified genetically related isolates in different groups, and the isolates were designated as Enterobacter cloacae strain GKE1(GenBank Accession Number: OP136122.1), Lysinibacillussp. strain GKL2 (GenBank Accession Number: OP133003.1), and Lysinibacillusfusiformis strain GKL1 (GenBank Accession Number: OP133000.1).