In this study, a biodegradable film from bacterial biopolymer, i.e., poly (3-hydroxybutyrate-co-4-hydroxybutyrate) or P(3HB-co-4HB) incorporating Thyme essential oil as an antimicrobial agent was developed as an active packaging for shelf-life extension of bread. Thin solvent-cast films containing 10, 20, and 30% v/w of Thyme oil were prepared and studied for the morphological, thermal, chemical, mechanical, barrier, and antimicrobial behaviors. The presence of oil in P(3HB-co-4HB) films and their purity without detectable remains of solvent, i.e., chloroform, was confirmed by infrared spectroscopy. In parallel, the time-bound release of volatile Thyme oil from the films was demonstrated when stored in a controlled environment. The plasticizing effect of Thyme oil in films was evidenced by decreased tensile strength and crystallinity. In contrast, an increase in elongation at break and water vapor permeability of oil films were measured. Finally, the antimicrobial activity of oil films was checked against total bread molds and observed that when bread was sealed in P(3HB-co-4HB) films containing 30% v/w Thyme oil, its shelf-life extended to at least 5 days compared to 1–4 days for the neat film. • Thyme oil completely inhibited the growth of bread molds than other essential oils. • P(3HB-co-4HB) films containing Thyme oil were successfully developed. • Thyme oil influenced the physicochemical and thermal properties of composite films. • Films showed antimicrobial activity against the total bread molds. • The film containing the 30% v/w Thyme oil extended the shelf-life of packed bread. [ABSTRACT FROM AUTHOR]