Antimicrobial activity of Jatropha curcas Linn. seedcake from biofuel production has been studied. For safety evaluation, mutagenic and cytotoxic test were carried out. The seedcake was extracted by maceration method with methanol. The antimicrobial activity, cytotoxicity test, and mutagenic effect were determined by disc diffusion, Brine Shrimp Lethality Test (BSLT), and Ames test method, respectively. The methanolic extract of J. curcas seedcake and fractions extract showed potent antimicrobial activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Candida albicans at minimum inhibitory concentration (MIC) of £ 1 mg/mL. The cytotoxicity value (LC₅₀) of methanolic extract of J. curcas seedcake was 22.12 µg/mL and had no mutagenicity effect against Salmonella typhimurium TA 1535. From this investigation it is evident that J. curcas seedacake from biofuel production might have promising biological activities including antimicrobial and cytotoxic properties. Therefore it could be a good source of natural medicine.