Pseudomonas aeruginosa are ubiquitous gamma-proteobacteria capable of producing the biosurfactant rhamnolipids (RL) and the polymer polyhydroxyalkanoate (PHA). RL are glycolipids with high biotechnological potential, whereas PHA is used for the production of biodegradable plastics. It has been proposed that the beta-oxidation pathway provides intermediates for RL biosynthesis, even when using a non-fatty acid carbon source for growth, while an intermediate of de novo fatty acid biosynthesis (FASII) pathway [(R)-3-hydroxyacyl-ACP] is used for PHA biosynthesis. The aim of this work is to study the inter-relationship of the RL and PHA biosynthetic pathways in a culture medium with a non-fatty acid carbon source, focusing on the role of FASII and beta-oxidation in supplying the substrates for the first step in RL and PHA synthesis, carried out by the RhlA and PhaG enzymes, respectively. The PHA synthases (PhaC1 and PhaC2) are only able to use CoA-linked 3-hydroxy acids and the PhaG enzyme catalyzes the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA, the substrate of PhaC1 and PhaC2. RhlA in turn catalyzes the synthesis of the RL precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) by the dimerization of two 3-hydroxyalkanoic acid molecules (that have been shown to be also (R)-3-hydroxyacyl-ACP). In this work, we show that RhlA can produce both RL and PHA precursors (presumably CoA-linked HAA), that the blockage of carbon flux through beta-oxidation pathway does not decrease RL titer, and that the enoyl-CoA hydratase RhlY and enoyl-CoA hydratase/isomerase RhlZ produce the main fatty acids precursor of RL using as substrate also a FASII intermediate (presumably (S)-3-hydroxyacyl-CoA).