This study evaluated ultrasound-assisted extraction and cold atmospheric plasma treatment for enhancing antioxidant and antibacterial activities in two phases. Phase I optimized ultrasound by varying power (0-200 W) and time (0-15 min) using a quadratic response surface methodology (RSM) to improve extraction yield and total phenolic content (TPC). Phase II compared ultrasound and cold plasma pretreatments (300 seconds), focusing on total flavonoid content (TFC), phytochemical composition (FTIR), and their antioxidant and antibacterial properties. The maximum extract yield (4.067%) and TPC (5.851 mg GAE/g DW) were achieved at 200 W for 15 minutes. Phytochemical analysis using FTIR and GC-MS identified linalool, β-pinene, cis-linalool oxide, trans-linalool oxide, pinocarveol, and camphor as the major compounds in the extract. The ultrasound-extracted samples showed significantly higher TFC (46.63 ± 4.01 mg QE/g) compared to cold plasma (32.83 ± 5.15 mg QE/g) and control (18.06 ± 2.48 mg QE/g). Antioxidant capacity, measured through DPPH inhibition, was also enhanced in ultrasound-treated extracts (47.8± 4.30%). The ultrasounic method exhibited superior antibacterial activity, particularly against Escherichia coli and Staphylococcus aureus. The minimum inhibitory concentration (MIC) was 12.5 μg/mL for S. aureus and 100 μg/mL for E. coli in plasma-treated samples. This study demonstrates that optimizing ultrasound and cold atmospheric plasma enhances the bioactive properties of A. millefolium, making it a promising technique for future applications in the food and pharmaceutical industries.