Chitosan (CS) as a biopolymer owes remarkable features; biocompatibility, biodegradability, antimicrobial properties, and non-toxicity making it an excellent candidate as an antibacterial agent applicable in food packaging. Despite all efforts that have explored chitosan properties, contradictory results are still found, specifically in describing antibacterial efficacy. Also, the uncertainty about chitosan's antibacterial mechanism acting against various microorganisms cannot clearly explain the observed conflicts. In this research, we have investigated the physiochemical and antibacterial properties of heat-treated chitosan cast films. The cast films were thermally treated for three weeks at 70 °C in a vacuum oven after being dried for 24 h in ambient conditions. The obtained chitosan cast films were characterized every seven days by FT-IR, XRD, and TGA to study the effect of heating. Also, the solubility and stability of the obtained films along with antibacterial activity were characterized in various media. It was found that the CS solubilisation step in acetic acid reduced the thermal stability and crystallinity of the cast films in comparison with the CS powder. But, interestingly, the heat treatment helped to recover the original properties of the “native” form. At the same time, the film stability was improved in aqueous solutions, leading to a reduction in the amount of chitosan released from the dried films estimated by a colorimetric technique and UV-vis absorbance. The antibacterial activity of CS films was evaluated against Paenibacillus, one of the main bacteria that causes milk spoilage. It was established that the released chitosan in its protonated form is responsible for the antibacterial performance of CS films. This subject hasn't been largely studied in the existing literature and the outcomes of this study may clarify some conflictual data observed in previous work.