Biofilm formation is one of the crucial factors for virulence in Candida albicans, Escherichia coli and their polymicrobial biofilm formed on medical prosthetics. In this study, high biofilm forming C. albicans M207 from a case of invasive candidiasis and a standard strain of bacterial culture E. coli ATCC 39936 were used for initial screening and media optimization using One Variable at a time (OVAT) and Response Surface Methodology (RSM) models, respectively, in polystyrene microtiter plates for in vitro biofilm studies. Sucrose and peptone significantly affected biofilm formation when screened by OVAT. Media optimization by Central Composite Design (CCD) showed that pH significantly affected biofilm formation for all the cultures which was further validated at critical parameters. Biofilm was quantified by growth OD measurements, MTT and CV assays in the presence and absence of FBS precoating and compared with standard Trypticase Soy Broth (TSB) media which did not show any significant difference. This high throughput polystyrene microtiter plate assay for preliminary screening of nutritional components using OVAT and media optimization using RSM model is being reported for the first time for biofilm formation by C. albicans and E. coli in a single and dual species environment. This study is essential for functional analysis of in‐vitro growth and biofilm formation by these two pathogenic strains on polymeric hospital equipments and to develop suitable control measures to inhibit biofilm formation by C. albicans and E. coli single species and their co‐culture.