Bidens pilosa L. is a species of plant that grows wild. It is commonly found in abundance in the Mekong Delta region of Vietnam. This study aimed to investigate the chemical composition, in vitro and in vivo antioxidant activity, the antimicrobial activity against some aquatic pathogenic bacteria and antifungal activity against plant pathogenic bacteria of Bidens pilosa L. extract. The results showed that Bidens pilosa L. extract had good antioxidant capacity through all four test methods of DPPH, ABTS●+, RP, and TAC with the EC50 values ​​of 455.78 ± 3.28 μg/mL, 148.68 ± 2.02 μg/mL, 462.09 ± 12.57 μg/mL and 139.14 ± 4.34 μg/mL, respectively. Fruit flies fed on a diet supplemented with 0.5 g/mL of Bidens pilosa L. extract had an average lifespan of 2.15 times and 1.54 times longer under oxidative stress using 20 mM Paraquat and H2O2 10%, respectively. In addition, the total polyphenols and total flavonoids in the extract were also determined to be 107.49 ± 4.04 mg GAE/g and 165.63 ± 2.90 mg QE/g, respectively. Regarding the antimicrobial activity, the ethanol extract of Bidens pilosa L. showed stronger resistance to Gram (+) S. agalactiae than the tested Gram (-) bacteria, including A. hydrophila, E. ictaluri, and A. dhakensis. In addition, the ethanol extract from Bidens pilosa L. also showed the better ability to inhibit the growth of the fungus Colletotrichum sp. (MIC = 1250 µg/mL) than that of Fusarium oxysporum (MIC = 2500 µg/mL).