Chemical composition and physico-chemical characteristics of natural and ex-situ reconstructed sea surface microlayer samples were studied using a complex methodological approach. Surface microlayer samples and the underlying seawater were collected in different seasons and different weather conditions in the northern most part of the Adriatic sea. The techniques used were thin layer chromatography with flame ionization detection for lipid classes analysis, electrochemical characterization of adsorbable organic substances using the ONP probe, and monolayer techniques for surface pressure (pi) and surface potential (Delta V) measurements, as well as Brewster angle microscopy. Our results indicated higher enrichment of organic matter for the microlayer sample collected in the warm period of the year (summer). This was reflected in the high surface activity of the present organic substances, and formation of a more condensed layer than for the other two samples collected in the spring. Of the two spring samples, the one sampled on a sunny day was reacher in surface active organic material. We concluded that the most important source of surface active substances is the in-situ production of organic susbtances by the present plankton and bacteria, promoted by sunlight, while antrophogenic input comprises a smaller part of the present organic matter, around 10% for all samples.