<p><strong>Objective</strong>. To isolate biosurfactant-producing microorganisms from the rhizosphere of fique and to select the best genus to evaluate the effect of nutritional and fermentation conditions on the production of rhamnolipids. <strong>Materials and methods</strong>. Rhizospheric soil was sampled in three areas of Cauca. The best genus was selected for the experimental designs (Plackett Burman and 2<sup>2</sup> factorial) and to find the production conditions for the growth kinetics at an Erlenmeyer flask scale. <strong>Results</strong>. Isolates from the rhizosphere of fique plants were from groups (or genera) of <em>Bacillus, Pseudomonas </em> and <em>Streptomyces</em>, being <em>Pseudomonas</em> the more responsive in preliminary testing for emulsification. From the results of the experimental designs and the kinetics of production, we found that rhamnose synthesis associated with rhamnolipids (3.2 g/l) and emulsification (68% EC24) was significantly favored (p <0.0001) by cultivating an inoculum of 10% v/v of <em>Pseudomonas fluorescens</em> in a medium composed of: soybean oil 2% (v/v), K<sub>2</sub>HPO<sub>4</sub> 0.2% (w/v), yeast extract 0.4 g/l, NH<sub>4</sub>NO<sub>3</sub> 3.7 g/l, 1 ml trace elements (CoCl<sub>3</sub> 20 mg/l, H<sub>3</sub>BO<sub>3</sub> 30 mg/l, ZnSO<sub>4</sub> 10 mg/l, Cu<sub>2</sub>SO<sub>4</sub> 1 mg/l, Na<sub>2</sub>MoO<sub>4</sub> 3 mg/l, FeSO<sub>4</sub> 10 mg/l MnSO<sub>4</sub> 2,6 mg/l) and pH 7.2. <strong>Conclusion</strong>. Of all the microbial genera isolated from the rhizosphere of fique, <em>Pseudomonas fluorescens</em> had the greatest potential in the production of biosurfactants of the rhamnolipids family.</p> <p><strong>Key words</strong>: <em>Pseudomonas fluorescens</em>, biosurfactant, rhamnose, emulsification index, soybean oil.</p><strong></strong> <p> </p>