Guava is consumed in natura and is also of considerable importance to the food industry. The seeds and peel of this fruit are discarded, however, guava seeds yield oil (~13%) that can be used for the bioproducts synthesis. The use of a by-product as a carbon source is advantageous, as it reduces the environmental impact of possible harmful materials to nature, while adding value to products. In addition, the use of untested substrates can bring new yield and characterization results. Thus, this research sought to study rhamnolipids (RLs) production from guava seed oil, a by-product of the fructorefinery. The experiments were carried out using Pseudomonas aeruginosa LBI 2A1 and experimental design was used to optimize the variables Carbon and Nitrogen concentration. Characterization of RLs produced occurred by LC-MS. In this study, variables in the quadratic forms and the interaction between them influenced the response (p < 0.05). The most significant variable was N concentration. Maximum RLs yield achieved 39.97 g/L, predominantly of mono-RL. Characterization analysis revealed 9 homologues including the presence of RhaC₁₀C_14:2 (m/z 555) whose structure has not previously been observed. This research showed that guava seed oil is an alternative potential carbon source for rhamnolipid production with rare rhamnolipid homologues.