Aims: In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays. Methods and Results: The assays were performed in microtitre plates with two minimal and two enriched broths, with one- or two-steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one- and two-steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathematical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm. Conclusions: Escherichia coli forms biofilms on static assays in a method-dependent fashion, depending on strain, and it is strongly modulated by culture conditions. Significance and Impact of the Study: As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, considering all variables in the experimental settings.