Antibacterial and antifungal activities of Chenopodium ambrosioides Linn have been studied for their medicinal potential by agar well diffusion method. Five bacterial strains, Escherichia coli, Klebsiella pneumoniae (Gram negative bacteria), Staphylococcus aureus, Bacillus subtilis and Staphylococcus epidermidis (Gram positive bacteria) as well as five fungal strains, Aspergillus niger, Aspergillus parasiticus, Trycophyton horzianum, Rhizopus tolenapur and Aspergillus flavus , were used to study the antimicrobial potential of the crude methanolic extract along with n-hexane, ethyl acetate, dichloromethane, n-butanol and aqueous fractions from the roots of Chenopodium ambrosioides Linn. The tested bacterial strains were taken from Center for Phytomedicine and Medicinal Organic Chemistry (CPMMOC) University of Peshawar, Pakistan; they were previously collected from hospital patients while the antifungal strains were collected from Center for Biotechnology and Microbiology (CBM) University of Peshawar, Pakistan which were also in advance collected from hostel patients of Khyber Teaching Hospital, University road Peshawar. The selected strains were tested against crude extract and its fractions. Zone of inhibition were measured by using National Committee for Clinical Lab Standards (NCCLS) method in which for antibacterial activities, Streptomycine while for antifungal activities, Miconazole were used as standard drugs. Dimethyl sulphoxide (DMSO) was used as negative control in both cases. All fractions remained inactive against K. pneumonia while other fractions showed good to non-significant activities against other bacterial strains. The n-hexane fraction showed moderate activity against A. niger while all other fractions showed low activity against antifungal strains. The Statistical Package for the Social Sciences (SPSS) calculations by using test statistics "t" shows the 'p' value of lower than α=0.05, while the confidence interval (CI), 95% is also significant.