Abstract A unique location with a unique environment attracted many researchers to explore bacteria as an isolate collection especially for actinomycetes group because of the potential of their antimicrobial metabolites. Information on antimicrobial activity by Streptomyces from the Sidoarjo mud area is limited. Biosurfactant was also one of the metabolites produced by the group of actinomycetes. Biosurfactant screening was carried out by growing isolates in a series of tests in the hemolytic, tributyrin agar, surface tension, and emulsification activity. Bacteria fermented on glycerol, n-hexane and molasses substrates for 7 days. The antimicrobial activity of organic extract evaluated using a paper disc diffusion method against E. coli ATCC 25922 and S. aureus ATCC 6538P. The results of biosurfactant screening tests showed differences in surface tension and varying emulsification activities in the isolates obtained. Supernatants produced by biosurfactants have been shown to be able to show antimicrobial activity as indicated by the formation of inhibitory zones associated with pathogenic growth. AF1 as a selected isolates identified as Streptomyces sp. MN394821 using 16S rDNA gene analysis. It has 100% similarity with accession-coded strain KY236015.1 and 98% with accession coded Streptomyces rochei AB1 strain GU434672.1.