Natural antimicrobial agents are the primary compounds incorporated into active packaging, offering a promising solution for enhancing food preservation and safety. This study aimed to develop potato starch films with varying concentrations of rosemary essential oil (REO) using the casting method. The films were evaluated for their structural, optical, mechanical, antimicrobial, and biodegradable properties, as well as the stability of their volatile compounds under varying storage times and temperatures. The most abundant volatile compounds identified were 1R-α-pinene, camphene, β-pinene, β-myrcene, and D-limonene, regardless of REO concentration or storage conditions. Generally, refrigeration was more effective in preserving a greater number of compounds at 5.0% and 7.5% REO concentrations. Incorporation of REO reduced the growth of Staphylococcus aureus by 92.9%, 94.5%, and 96.2%, and Escherichia coli by 96.5%, 98.0%, and 99.0%, respectively, compared to the control. The essential oil also exhibited more antimicrobial activity against S. aureus. The addition of REO reduced film thickness while increasing elongation and water solubility without affecting moisture content or water vapor permeability. Biodegradability tests confirmed the environmentally friendly nature of the films. This study demonstrates the potential application of these films as active and biodegradable packaging for food storage under various temperature conditions.