In marine environments, biofilm can cause negative impacts, including the biofouling process. In the search for new non-toxic formulations that inhibit biofilm, biosurfactants (BS) produced by the genus Bacillus have demonstrated considerable potential. To elucidate the changes that BS from B. niabensis promote in growth inhibition and biofilm formation, this research performed a nuclear magnetic resonance (NMR) metabolomic profile analysis to compare the metabolic differences between planktonic cells and biofilms of Pseudomonas stutzeri, a pioneer fouling bacteria. The multivariate analysis showed a clear separation between groups with a higher concentration of metabolites in the biofilm than in planktonic cells of P. stutzeri. When planktonic and biofilm stages were treated with BS, some differences were found among them. In planktonic cells, the addition of BS had a minor effect on growth inhibition, but at a metabolic level, NADP+, trehalose, acetone, glucose, and betaine were up-regulated in response to osmotic stress. When the biofilm was treated with the BS, a clear inhibition was observed and metabolites such as glucose, acetic acid, histidine, lactic acid, phenylalanine, uracil, and NADP+ were also up-regulated, while trehalose and histamine were down-regulated in response to the antibacterial effect of the BS.