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Escherichia coli has carved its niche in the urinary tract with the formation of a formidable matrix called biofilm. This biofilm is not only recalcitrant to the body’s immune system but also resistant to antibacterial agents. Senna siamea (Lam) Irwin and Barneby is a medicinal plant with established antibacterial effect against planktonic cells of many bacteria. An attempt was made herein to evaluate the effect of its leaf extract and fractions on biofilm of E. coli isolates. Crude extracts of leaf, stem bark and root of this plant were prepared using ethanol as the solvent for the cold extraction. Phytochemical screening was carried out on the three extracts. Two E. coli strains from different antenatal patients attending General Hospital, Kafanchan, Kaduna were donated to us by a researcher from Ahmadu Bello University, Zaria and the reference strain, E. coli, WDCM 00013 (from Germany) were tested for biofilm production using the Congo red method. Antimicrobial susceptibility testing of the crude extracts against the isolates was carried out using the agar diffusion method. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) were determined for the leaf extract of the plant using micro broth dilution and agar diffusion methods respectively. In order to establish the antibiofilm activities of the leaf extract of the plant, sub-inhibitory concentrations (sub-MIC) were used against the test isolates in the remaining assays in the work. Column chromatography backed by thin layer chromatography (TLC) was used to fractionate leaf extract (having the best antibacterial activity) of the plant, using different ratios of a combination of hexane, ethyl acetate and n-butanol as fractionating solvents. MIC and MBC of the leaf extract were and 50 mg/ml respectively. High values of percentage biofilm inhibition were observed against all the bacterial isolates from the antibiofilm assay. Combination of solvents in the increasing order of polarity enhanced the antibiofilm activity of the various fractions of the leaf extract of Senna siamea. In conclusion, further fractionation of Senna siamea leaf extract increases its antibiofilm activities.