Arachidate Langmuir-Blodgett (LB) films of different chemical composition and number of monomolecular layers deposited on silylated silica glass substrates were studied by means of positron annihilation spectroscopy. The applied methods included the measurement of the Doppler broadening of the annihilation photopeak with variable energy positron beams and bulk positron lifetime measurements. The studied samples were 58 monomolecular layers (MML) thick Mg- and Cd-arachidate, arachidic acid (50 MML) and a series of Pb-arachidate samples with 4, 10, 20, 40, and 58 MML. The investigation showed that the variable energy positron beam technique is capable of measuring the thickness of the deposited LB films. The measured positron annihilation parameters are sensitive to the chemical composition of the films and the behavior of the films in a vacuum. The results confirmed the stability of salt base LB films in high vacuum conditions and showed the desorption of pure acid films. These investigations have also shown that a strong positron trap is formed in the near-surface region of the hydrophobized substrate as a consequence of the silylation process. The results suggest that positron beams provide valuable complementary information to results obtained by other techniques.