The main objective of this study was to isolate compounds from roots of Sida rhombifolia and subsequently evaluate their antibacterial activities. Crude gradient extracts were obtained from three solvents (petroleum ether, chloroform and methanol) with increasing solvent polarity using cold maceration technique. The in vitro antibacterial activity evaluation of gradient extracts and isolated compounds was done on four different pathogenic bacterial strains (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium) using agar disc diffusion technique. The results showed that antibacterial activities were comparable to each other. But their activities were relatively weaker as compared to that of the reference compound (ciprofloxacin). Among the three crude extracts, the chloroform extract was subjected to column chromatographic separation that led to isolation of SRL-1, SRL-2 and SRL-3. The chemical structures of the compounds were found to be n-hexacos-11-enoic acid, stigmasterol and β-sitosterol, respectively, based on physical properties and spectroscopic (IR and NMR) data as well as literature reports. The observed antibacterial activities of the crude extracts and the isolated compounds could justify the traditional use of the plant for the treatment of different bacterial infections. Thus, further test is recommended on large number of bacterial strains to decide the potentials of the compounds as candidates in development of antibacterial drugs.