Objective: To study the antibacterial and antifungal activities of methanolic extract of roots of Carissa opaca and its fractions in hexane, chloroform, ethyl acetate, n-butanol and water, and the isolated compounds. Methods: The zones of inhibition of the samples against test microorganisms were determined by agar well diffusion method. Minimum inhibitory concentrations of the samples were determined by agar well dilution method. Test microorganisms included four standard bacteria [Bacillus subtilis ATCC 6633 (B. subtilis), Escherichia coli ATCC 8739 (E. coli), Pseudomonas aeruginosa ATCC 9027 (P. aeruginosa), and Staphylococcus aureus ATCC 6538], two standard fungi [Candida albicans ATCC 10231 (C. albicans)] and Aspergillus niger, and six clinical isolates (B. subtilis, E. coli, P. aeruginosa, Staphylococcus aureus, Salmonella typhi and Enterobacter cloacae). The most active fraction was investigated to isolate compounds. The chemical compounds isolated from the ethyl acetate fraction were identified by gas chromatography-mass spectrometer, high performance liquid chromatography and liquid chromatographymass spectrometer. Results: E. coli, P. aeruginosa, and C. albicans were the most susceptible. Less polar fractions exhibited stronger efficacy than polar ones, and ethyl acetate fraction proved to be the most potent. Zones of inhibition of hexane, chloroform and ethyl acetate fractions, and amoxil against C. albicans were 19.96, 22.01, 23.10 and 19.20 mm, respectively. Ethyl acetate faction was the most toxic to all the test microorganisms, with minimum inhibitory concentrations of 8.0, 7.8 and 7.78 μg/mL against P. aeruginosa, C. albicans and B. subtilis, respectively. Isolated compounds, limonene, 2'-hydroxyacetophenone, vanillin, naphthalenone, 2,3,3-trimethyl-2-(3-methylbuta-1,3-dienyl)-6-methylenecyclohexanone, 2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester, β-sitosterol, vitamin E, rutin, quercetin, lupeol, epigallocatechin, showed considerable antimicrobial activities against test microorganisms. Conclusions: The roots of Carissa opaca contain compounds with significant antimicrobial potential.