Jatropha gossypifolia L., popularly known as the belly-ache bush is a rich natural source of novel macrocyclic diterpenoids. Crude root and stem extracts of J. gossypifolia were tested against certain human bacterial pathogens. Among the treatments, lipophilic hexane extract of the dried roots inhibited the gram negative bacterial human pathogens Corynebacterium diphtheriae, Bacillus cereus and Proteus mirabilis. Effective inhibition (18 mm) was observed against C. diphtheriae, the activity being dose-dependent among the dilutions tested, with maximum efficacy at 500 ppm. B. cereus was resistant at lower dilutions (25-100 ppm), however, 250 and 500 ppm concentrations exhibited effective inhibition (8.5 and 9.1 mm). Marginal inhibition was observed against P. mirabilis (5.5 mm) at 500 ppm. Of the 7 column fractions tested, fraction-7 exhibited significant activity against C. diphtheriae, B. cereus, P. mirabilis, Shigella flexneri, Klebsiella aerogenes and Staphylococcus aureus. Gram-negative organisms tested, except P. mirabilis and S. flexneri, showed marginal inhibition for the crude extracts. Crystalline fraction-7 appreciably inhibited K. aerogenes and S. aureus at higher concentrations (1000, 500 and 250 ppm). X-Ray Diffraction Crystallography (XRD) analysis of the crystals confirmed that it is the known macrocyclic diterpenoid, Jatrophone, with a few other proven biological activities.