Aims: Bidens pilosa is an extraordinary source of phytochemicals particularly flavonoids especially in leaves which have been attributed in various studies due to its antibacterial properties. The present study aimed at addressing bio-burden of chronic wound through proving a possible source of new antimicrobial product for wound healing. Methodology: Solvent-solvent extraction method was used to isolate crude flavonoid fraction from leaves of B.  pilosa using ether, chloroform, ethylacetate and methanol (1:1:1). Thin-layer chromatography was used to identify crude flavonoid fraction using methanol/n-hexane (1:2: v/v) as mobile phase solvents. Agar well diffusion method was used to determine anti-bacterial activity of crude flavonoid against bacterial pathogens: Susceptible Pseudomonas aeruginosa ATCC®27853™, resistant Pseudomonas aeruginosa susceptible Staphylococcus aureus ATCC®25923™, methicillin resistant Staphylococcus aureus, Streptococcus pneumoniae and methicillin resistant Staphylococcus epidermidis. Minimum inhibitory concentration (MIC) and Minimum bactericidal contrition (MBC) were also determined using broth dilution and culture methods. Results: Thin-layer chromatographic profiling revealed an identity of three different spots with flavonoids from B. pilosa leaves showing three bands with Rf values 0.51, 0.60 and 0.63. The mean and standard deviation zone of inhibition of crude flavonoids ranged from 11.50±0.50 mm to 17.50±1.50 mm. The mean and standard deviation of positive controls (Ciproflaxacin, Co-Amoxiclay and Voncomycin) ranged from 0.00±0.00 to 22.50±0.50 mm. MIC and MBC of crude flavonoids ranged from 12.5-25.0 mg/mL and 50 to 200 mg/mL respectively. The flavonoid fraction was more effective against gram positive bacteria than on gram negative bacteria and it exhibited bactericidal effect on Methicillin resistant Staphylococcus aureus, resistant P. aureginosa, sensitive P.aureginosa and S. pneumonia. Conclusion: B. pilosa leaves could be a potential source for future drug development from flavonoid to address the issue of need for new antibiotics due to alarming burden of antimicrobial resistance in last resort antibiotics.