Introduction: Andrographis paniculata is a plant employed in traditional medicine among the natives of Owerri, Eastern Nigeria, in the management of several ailments. In this study the leaf was investigated for its chemical constituents and to validate its antimicrobial activity in ethnomedicine. Methods: The fresh leaf of the plant was steam-distilled to obtain the volatile oil which was subjected to FT-IR and GC-MS analysis. The powdered air-dried leaf was also extracted with methanol to obtain the crude extract which was subjected to phytochemical analysis. The crude methanolic extract was also partitioned into hexane, chloroform, ethylacetate and 50% methanol in chloroform to obtain the corresponding fractions. The crude extract and fractions were tested for activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus niger. The biologically active chloroform fraction was further purified using column chromatography and the fractions subjected to GC-MS analysis. Results: The steam distillation gave an oil and the GC-MS analysis indicated the presence of butylated hydroxytoluene, hexadecanoic acid, methyl ester, dibutyl phthalate, 8,11-octadecadienoic acid, methyl ester, 11-octadecenoic acid, methyl ester and bis (2-ethylhexyl) phthalate. Phytochemical analysis of the crude extract showed the presence of tannins, alkaloids, sterols/steroids, flavonoids, volatile oils, terpenoids, saponins and phenols. Among the extractives, the chloroform-soluble fraction showed the highest activity against S. aureus and B. subtilis while none of the extracts was active against C. albicans and A. niger. It was also found that the hexane-soluble fraction was inactive against all the test organisms while the ethylacetate-soluble fraction showed the broadest spectrum of anti-bacterial activity. Column chromatographic separation of the biologically active chloroform-soluble fraction gave fractions which on GC-MS analysis and by comparison with the standard library computer MS data led to the identification of some chemical constituents, including aromatic compounds, sterols, alicyclics and long-chain hydrocarbons and carbonyl compounds such as carboxylic acids, esters and lactones. Conclusion: The antimicrobial activity exhibited by the leaf extracts support the traditional medicinal use of the leaf to manage infections. Also, the presence of the identified phyto-constituents may be responsible for the ethno-medicinal properties of the plant.