<p>In present study, defatted watermelon seeds flour (DWMSF) based film forming solution and active films functionalized with oregano essential oils was developed using ultrasonication treatment (time – 40, 50 &amp; 80&#xa0;min; amplitude- 20%, 30% &amp; 40%). The findings suggested that the increasing time and amplitude of ultrasonic treatments, significant (<i>p</i> &lt; 0.05) improved the physical, mechanical, barrier, thermal, and optical properties of films. The lowest particle size (4.79&#xa0;μm), zeta potential (-21.7&#xa0;mV), and decreased viscosity (flow behavior index, <i>n</i> = 0.094) were recorded in the ultrasonicated (40% amplitude for 80&#xa0;min) film formulation (WM 40:80) compared to the untreated control sample (WM 0:0). Similarly, the thickness (0.25&#xa0;mm), swelling degree (0.05%), and opacity (0.41%) were lowest in the (WM 40:80) samples in comparison to control (WM 0:0) samples. The treated active packaging films showed a lower oxygen transmission rate (WM 40:80 = 8.12 cm³/m²d) than the control films (WM 0:0 = 18.10 cm³/m²d). The microstructural (SEM) analysis showed a uniformly distributed polymeric matrix with a smooth, flat surface, and declined crystallinity in the treated film (WM 40:80) compared to the control film (WM 0:0). Combining ultrasonication treatment and the incorporation of OEO improved the antimicrobial efficiency of DWMSF-based active packaging films.</p> Graphical abstract <p></p>