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Rhamnolipid biosurfactant produced by Pseudomonas aeruginosa, possesses non-toxicity, environmental compatibility, a wide range of pH (4–8), temperature (4–100 °C), and salinity (1–10%) stability. The application of RLs is worldwide accepted in the pharmaceutical, medicinal, and food industries. It has been used for cytotoxicity efficacy analysis with a limited number of cancerous cell lines. To widen the scope of rhamnolipid application as an anticancer agent, we have studied Di-RLs homolog, ‘Rha-Rha-C10-C10’ produced by Pseudomonas aeruginosa RA5 against human cancerous cell lines including breast cancer (MCF-7), leukemia (K-562), cervical cancer (HeLa), Lung cancer (HOP-62), and colon cancer (HT-29) in a dose-dependent way. It was purified with silica gel chromatography followed by TLC and mass spectroscopy prior to cytotoxicity analysis. With a tensiometer, critical micelle concentration of …