New chemical structures with antifungal properties are highly desirable from the point of view of modern pharmaceutical science, especially due to the increasingly widespread instances of drug resistance in the case of these diseases. One way to solve this problem is to use polymeric drugs, widely described as biocidal, positively charged macromolecules. In this work, we present the synthesis of new cationic β-glucan derivatives that show selective antifungal activity and at the same time low toxicity toward animal and human cells. Two β-glucans isolated from oats and barley and modified using glycidyltrimethylammonium chloride were obtained and evaluated for biocidal properties on the cells of mammals and pathogenic fungi and bacteria. These compounds were found to be nontoxic to fibroblast and bacterial cells but showed selective toxicity to certain species of filamentous fungi (Scopulariopsis brevicaulis) and yeasts (Cryptococcus neoformans). The most important aspect of this work is the attempt to explain the mechanisms of action of these compounds by studying their interaction with biological membranes. This was achieved by examining the interactions with model biological membranes representative of given families of microorganisms using Langmuir monolayers. The data obtained partly show correlations between the results for model systems and biological experiments and allow indicating that the selective antifungal activity of cationic β-glucans is related to their interaction with fungal biological membranes and partly lack of such interaction toward cells of other organisms. In addition, the obtained macromolecules were characterized by spectral methods (Fourier transform infrared (FTIR) and 1 H nuclear magnetic resonance (NMR) spectroscopies) to confirm that the desired structure was obtained, and their degree of modification and molecular weights were determined.