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Bacterial biofilms are associated with a large number of persistent and chronic infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics and immune defenses, which makes it hard to eradicate biofilm-associated infections. This study aimed to analyze the effect of zinc sulphate (ZnSO4) and bacteriophage treatment on E. coli biofilm. This study was performed in microbial biotechnology laboratory, SUST, Sylhet, Bangladesh during a period of 9 months from December 2016 to August 2017. Bacteria were isolated from clinical samples of renowned hospital and diagnostic center in Sylhet city, Bangladesh and identified using various biochemical tests. Biofilm forming isolates were screened by the crystal violet assay method in microtiter plate. Bacteriophage was isolated from drainage samples and recognized by spot test. Biofilm enhancement was studied using different concentrations of FeCl3. Also, Biofilm inhibition was investigated using zinc sulphate (ZnSO4) and bacteriophage separately and in combination by the enumeration of viable cells of biofilm after treatment in six (6) days experiment. Kirby-Bauer disk diffusion method was used for antibiotic susceptibility test. The result showed that biofilm formation was enhanced by the supplementation of 10 µM FeCl3 in medium. A significant decrease (p ˂0.01) in viable cells of biofilm was occurred with highest reduction of 89±1% and 85.38±1.30% by zinc sulphate and phage treatment, respectively. Also, the combined use of zinc sulphate and phage caused significant removal (p ˂0.01) in viable cells of biofilm with highest reduction of 97.22±1.37% in comparison to control. However, there was significant difference (p <0.01) between combined treatment and single treatment. The antibiotic susceptibility test showed that selected biofilm forming isolate was resistant to antibiotics tested. Finally, it might be said that a combined approach of bacteriophage and iron antagonizing molecule is more significant than distinct approach for the eradication of E. coli biofilm.