Pseudomonas aeruginosa PAO1-B13 isolated from Cooktops of kitchen stove can produce amphipathic compounds, which allows it to perform some surface activities and are widely named rhamnolipid biosurfactant. Here, we report promising antimicrobial activity of rhamnolipid produced by the above strain against some tested fungi and bacteria in addition to antitumor activity against both Breast and Colon Carcinoma cell lines. Also, Rhamnolipid exhibited a high recovery of heavy metals from suspensions in the field of bioremediation. The cluster genes encoding for biosurfactant production (rhlB, rhlC and rhlI) were isolated and sequenced successfully. For these promising properties, efforts have been made for maximizing the production of rhamnolipid for commercial use through optimization of various nutritional and environmental factors. The maximum production rate was achieved at 37 ˚C, pH 6.8, incubation period for one week at 150 rpm, only one carbon source; sunflower oil; inoculum size, 4.84 x106 CFU; amino acid, Dl-valin; KCl, 500 ppm; vitamins, none. CH3Cl:CH3OH was used to extract Rhamnolipid in a 2:1 ratio. Here, we recommend further investigation for this promising strain for large-scale use in the field of pharmaceutical and bioremediation.