In this study, Rhodamine B-containing chitosan-based films were prepared and characterized using their mechanical, photophysical, and antibacterial properties. The films were synthesized using the casting method and their mechanical properties, such as tensile strength and elongation at break, were found to be dependent on the chemical composition and drying process. Infrared spectroscopy and X-ray diffraction analysis were used to examine the chemical structure and degree of structural perfection of the films. The photophysical properties of the films, including absorption spectra, fluorescence detection, emission quantum yields, and lifetimes of excited states, were studied in detail. Rhodamine B-containing films exhibited higher temperature sensitivity and showed potential as fluorescent temperature sensors in the physiological range. The antibacterial activity of the films was tested against Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli, with Rhodamine B-containing films demonstrating more pronounced antibacterial activity compared to blank films. The findings suggest that the elaborated chitosan-based films, particularly those containing Rhodamine B can be of interest for further research regarding their application in various fields such as clinical practice, the food industry, and agriculture due to their mechanical, photophysical, and antibacterial properties.