Objectives One of objectives of this study was to examine the intrinsic antimicrobial activity of the chitosan hydrogels, which were used as vehicles (bases) for incorporation of the azithromycin liposomes. To determine whether chitosan hydrogels could potentiate antibacterial activity of the incorporated azithromycin liposomes and to examine the effect of bilayer fluidity on the antibacterial activity of encapsulated azithromycin, the studies were performed with chitosan hydrogels containing incorporated azithromycin liposomes and the results were compared with azithromycin liposomes (liquid dispersions), solution of azithromycin, hydrogels containing azithromycin solution and the plain chitosan hydrogels. Material and Methods The experimental part of this study included preparation of the plain chitosan hydrogels from the different molecular weight chitosans as well as preparation and characterization of the several types of liposomes encapsulating azithromycin, which were incorporated into the chitosan hydrogels (liposomal hydrogels). Intrinsic antimicrobial activities of the plain chitosan hydrogels were examined by the reduction of number of the test microorganisms (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus brasiliensis), which were inoculated into the hydrogels, stored for 28 days and tested at determined time intervals. In vitro antibacterial activity of the different azithromycin containing liposomal hydrogels toward Staphylococcus aureus ATCC 6538 was performed by agar well diffusion method. Results All the plain chitosan hydrogels have shown intrinsic antimicrobial activities, confirming that there is no need for the use of preservatives in the topical formulations based on the chitosan hydrogels. Furthermore, the elasticities/rigidities of the different azithromycin liposomes have influenced their antibacterial activities as well as antibacterial activities of the liposomal hydrogels. Conventional liposomes, characterized by rigid bilayers, have demonstrated the most pronounced antibacterial activity. All the chitosan hydrogels contributed the antibacterial activity of the incorporated azithromycin liposomes, what was confirmed by a larger inhibition zones formed, compared to those obtained by hydrogels containing azithromycin solution and the zones formed by the free drug. Conclusion Considering all the results, it can be assumed that the chitosan hydrogels could be used as vehicles for topical formulations and liposomes aimed for the treatment of skin infections. However, further in vivo studies should be carried out to confirm the efficacy of the innovative formulations.