Medicinal plants are rich in bioactive compounds that can serve as precursors for the synthesis of alternative agents with minimal side effects, as opposed to synthetic drugs. Many microorganisms have developed resistance to available antibiotics; hence, there is a need to search for alternative antimicrobial agents from plants. This study aimed to extract and characterize essential oil from Tithonia diversifolia leaves and to evaluate its antimicrobial and antioxidant properties. The Oil was extracted by hydro-distillation and characterized by gas chromatography-mass spectrometry (GC-MS). Antimicrobial activity was assessed by the disc diffusion method, and antioxidant potential was evaluated by the DPPH method. The plant material yielded 10 mL of oil (0.66%), GC-MS analysis identified 43 compounds, with Norborneol (16.52%) as the most abundant. The extract had the highest inhibitory activity on Proteus mirabilis (43.00 mm) at a concentration of 100 mg/ml compared with gentamicin (reference standard) (34.00 mm). The inhibitory effects showed a dose-dependent pattern, as observed through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. Antioxidant analysis revealed the oil’s IC50 value as 0.087 µg/mL, compared to 0.024 µg/mL for the standard Vitamin C, indicating moderate antioxidant potential. In Conclusion, the essential oil from Tithonia diversifolia is highly promising as a natural agent for managing microbial infections and oxidative stress-related conditions due to its bioactive components.