Abstract: Background: The use of antibiotics over the years has led to increased bacterial resistance. With the current menace of microbial resistance to already existing antibiotics, there is an urgent need to discover new antimicrobial agents with different and novel mechanism of actions for new and re-emerging infectious diseases. Pupalia lappacea (L.) Juss is an annual or perennial herb (Family Amaranthaceae) which is used for the treatment of boils, wounds and skin infections. Objective: To determine the antimicrobial potential and antibiotic resistance modulation of the chloroform (PLC), petroleum ether (PLP) and ethanol (PLE) extracts and evaluate their possible cytotoxic effects. Methods: These include the determination of Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) Minimum Fungicidal Concentration (MFC) and microbial time-kill kinetics of the extracts against Pseudomonas aeruginosa ATCC 4853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis NTCC 10073, Proteus vulgaris ATCC 4175 and clinical strains of Streptococcus pyogenes, Klebsiella pneumonia and Candida albicans. The ability of the extracts at sub-MIC concentrations to modulate the activity of some selected antibiotics including amoxicillin, cloxacillin, tetracycline, erythromycin and ampicillin was evaluated. Influence of the extracts on viability, proliferation and Lactate Dehydrogenase (LDH) release of HaCaT keratinocyte cells were evaluated at various selected concentrations. Results: All the extracts of P. lappacea exhibited broad-spectrum antimicrobial activity. The PLC demonstrated the highest antimicrobial activity with MIC of 2.5 to 4.0 mg mL-1 and MBC/MFC value ranging from 4.0-8.0 mg mL-1. Time-kill kinetics generally revealed a concentration and time-dependent rate of microbiocidal activity of the extracts and MBC/MIC ratio <4. The PLC and PLP at sub-MIC concentrations of 0.5 mg mL-1 caused a 20% reduction in the MICs of amoxicillin, erythromycin, ampicillin and cloxacillin against K. pneumonia. There was also a 96% reduction in the MIC of amoxicillin against E. coli and P. vulgaris. None of the extracts exhibited cytotoxic activity against HaCaT keratinocytes within the concentrations used. Conclusion: All extracts of P. lappacea exhibited broad-spectrum antimicrobial activity and sub-MIC concentrations of the extracts showed microbial resistance modulation potential among the selected antibiotics.