AbstractPseudomonas aeruginosa 47T2, grown in submerged culture with waste frying oil as a carbon source, produced a mixture of rhamnolipids with surface activity. Up to 11 rhamnolipid homologs (Rha‐Rha‐C8‐C10; Rha‐C10‐C8/Rha‐C8‐C10;Rha‐Rha‐C8‐C12:1; Rha‐Rha‐C10‐C10; Rha‐Rha‐C10‐C12:1; Rha‐C10‐C10; Rha‐Rha‐C10‐C12/Rha‐Rha‐C12‐C10; Rha‐C10‐C12:1/Rha‐C12:1‐C10; Rha‐Rha‐C12:1‐C12; Rha‐Rha‐C10‐C14:1; Rha‐C10‐C12/Rha‐C12‐C10) were isolated from cultures of P. aeruginosa 47T2 from waste frying oil and identified by HPLC‐MS analysis. This article deals with the production, isolation, and chemical characterization of the rhamnolipid mixture RL47T2. The physicochemical and biological properties of RL47T2 as a new product were also studied. Its surface tension decreased to 32.8 mN/m; and the interfacial tension against kerosene to 1 mN/m. The critical micellar concentration for RL47T2 was 108.8 mg/mL. The product showed excellent antimicrobial properties. Antimicrobial activity was evaluated according to the minimum inhibitory concentration (MIC), the lowest concentration of an antimicrobial agent that inhibits development of visible microbial growth. Low MIC values were found for bacteria Serratia marcescens (4 μg/mL), Enterobacter aerogenes (8 μg/mL), Klebsiella pneumoniae (0.5 μg/mL), Staphylococcus aureus and Staphylococcus epidermidis (32 μg/mL), Bacillus subtilis (16 μg/mL), and phytopathogenic fungal species: Chaetonium globosum (64 μg/mL), Penicillium funiculosum (16 μg/mL), Gliocadium virens (32 μg/mL) and Fusarium solani (75 μg/mL). © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 316–322, 2003.