The aim of the research was to determine the influence of chitosan type and propolis extract concentration on biological and physicochemical properties of chitosan-propolis films in terms of their applicability in food packaging. The films were prepared using three types of chitosan: from crab shells, medium and high molecular weight and propolis concentration in the range of 0.75–5.0%. The prepared polysaccharide films were tested for antimicrobial properties, oxygen transmission rate (OTR) and water vapor transmission rate (WVTR). Moreover, sorption tests and structural analysis were carried out. Microbiological tests indicated the best antimicrobial activity for the film consisting of high molecular weight chitosan and 5.0% propolis extract. Both the type of chitosan and propolis concentration affected transmission parameters—OTR and WVTR. The best barrier properties were recorded for the film composed of high molecular weight chitosan and 5.0% propolis extract. The results of sorption experiments showed a slight influence of chitosan type and a significant effect of propolis extract concentration on equilibrium moisture content of tested films. Moreover, propolis extract concentration affected monolayer water capacity (Mm) estimated using the Guggenheim, Anderson and de Boer (GAB) sorption model. The obtained results indicate that chitosan films with an addition of propolis extract are promising materials for food packaging applications, including food containing probiotic microorganisms.