The phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme that interacts with membranes to catalyse phosphatidylcholine (PC) transformation. In this work, we focused on the interaction between PLDSc and two lipid activators: a neutral lipid, diacylglycerol (DAG), and an anionic one, phosphatidic acid (PA). DAG is a naturally occurring alcohol, so it is a potent nucleophile for the transphosphatidylation reaction catalysed by PLD. Concerning PA, it is a widely described activator of PLDSc-catalysed hydrolysis of PC. The monolayer technique allowed us to define PLDSc interaction with DAG and PA. In the case of DAG, the results suggest an insertion of PLDSc within the acyl chains of the lipid with an exclusion pressure of approximately 45 mN/m. PLDSc-DAG interaction seemed to occur preferentially with the lipid in the liquid-expanded (LE) phase. PLDSc interaction with PA was found to be more effective at high surface pressures. The overall results obtained with PA show a preferential interaction of the protein with condensed PA domains. No exclusion pressure could be found for PLDSc-PA interaction indicating only superficial interaction with the polar head of this lipid. Brewster angle microscopy (BAM) images were acquired in order to confirm these results and to visualise the patterns induced by PLDSc adsorption.