Gentamicin possesses strong adverse actions like oto and nephrotoxicity. The latter is a result of strong gentamicin-acid phospholipid interactions, resulting in cell fusion, fission, etc., ions as calcium interact with gentamicin and effectively deter its toxicity. In this work, the interactions of gentamicin and Ca(2+) with phosphatidylserine/phosphatidylcholine (PS/PC) mixtures of different ratio are experimentally characterized. Special attention is paid to bridge thermodynamic and morphological properties of adsorption monolayers and thin liquid films (TLFs) composed of these lipid mixtures. Our results show that gentamicin decreases the stability of common black TLFs formed of pure PS coupled with suppression of lipid surface adsorption to the monolayers at the air-water interface; also, gentamicin reveals effects of lowering of lipid spreading on the interface and significant loss of material during monolayer cycling, increase of condensed phase, and organization of dense net-like domain monolayer texture. Gentamicin addition results in opposite effects for films formed of DPPC/PS (95:5) mixture. It increases the stability of Newton black TLFs formed by DPPC/PS correlated with faster and stronger surface adsorption and better surface spreading; also, gentamicin lowers the amount of condensed phase and organization of domains of smaller size. We also showed that Ca(2+) itself decreases the stability of common black TLFs formed of PS accompanied with weaker surface adsorption, formation of higher amounts of condensed phase and organization of domains. In our experiments, Ca(2+) softens, even deters, the effects of gentamicin on both PS and DPPC/PS films.