Cucumis ficifolius (family Cucurbitaceae) is traditionally used for the treatment of diarrhea, teeth-ach, wound, burns, bleeding from external cuts and against different infectious diseases. With the absence of previous scientific report on the roots of the plant, this study was focused on isolation of chemical constituents and evaluation of their anti-bacterial activities of the roots of C. ficifolius. The n-hexane, ethyl acetate and methanol extracts of the roots of C. ficifolius were investigated for their phytochemical constituents and the results revealed that C. ficifolius is rich in many secondary metabolites such as saponins, terpenoids, glycosides, and steroids. But tannins and alkaloids were not observed. The ethyl acetate extract was subjected to column chromatography over silica gel and furnished three compounds namely compound-1, compound- 2 and compound-3. The structures of these compounds were characterized using FTIR, 1H-NMR, 13C-NMR and DEPT-135. The crude extracts and the isolated compounds were tested against four bacteria strains (Gram negative, E. coli and p. aeruginosa; Gram positive, S. aureus and B. subtilis) using Agar well diffusion method, the results showed that the methanol extracts and compound-2 were active against all the tested bacteria. Accordingly, 12.34, 18.25 and 23.75 mm zone of inhibition against B. subtilis were found at the concentration of 20, 30 and 40 μg/mL MeOH extract respectively. Similarly 9.5, 13.25 and 17.25 mm zone of inhibition were found for the same bacteria strains and concentrations of compound-2. These results showed the increase of inhibition zone with concentration. However, the n-hexane crude extract was found not to have anti-bacterial activity towards the tested bacteria strains.