Biofilm is an important strategy used by some microorganisms for survival. The present study was carried out to optimize the invitro culture conditions required for biofilm formation of Escherichia coli O157:H7. Tryptic soya broth (TSB) was employed as the growth medium for biofilm production using chitin flakes as inert surface. The 0.08 percent with 0.3 percent w/v chitin flakes optimum concentration of TSB required for maximum biofilm production by Escherichia coli O157:H7. Biofilm cell population reached peak on day two, while planktonic cell population started decreasing after day one post inoculation. Decline of biofilm cells was gradual compared to planktonic cells.