Waltheria indica Linn is a plant used for medicinal purposes to treat several diseases like cough, diarrhea, fever, hemorrhage, malaria, stomach ache and wounds. In this study, two compounds were isolated from the plant leaves and roots solvent fractions using chromatographic techniques. The antimicrobial activities of the compounds were evaluated on clinical microbial test strains associated with respiratory diseases. The chromatographic elution revealed two previously known compounds from the polar fractions of ethanol extract of leaf and the ethyl acetate fractions of the roots. The compounds were identified based on spectral analyses (IR, 1HNMR and 13CNMR) and physical characteristics as well as reported literature to be n- tetraicosanol and stigmasterol. The antimicrobial evaluation of the compounds using well diffusion method against eight pathogens of Gram positive/negative bacteria and fungi such as (S. aureus, S.pneumonia, S.pyrogens and K.pneumonia) and fungi strains (C.ulcerans, C.albicans, C. krusei and C. tropicalis) revealed susceptibility against all the test organisms, showing inhibition zones ranging from 27 -31 mm for n- tetraicosanol and 28-35 mm for stigmasterol against the test strains except against Streptococcus pyrogens and Candida albicans. However, the compounds were less active than the reference drugs, ciprofloxacin and fluconazole. This is the first report for the presence of stigmasterol and tetraicosanol from Waltheria indica plant. These two antimicrobial compounds confirm the ethno-medicinal use of the plant as an anti-infective agent and may serve as leads in the development of new antibiotics.