Microbial life is predominantly observed as biofilms, which are a sessile aggregation of microbial cells formed in response to stress conditions. The microtiter dish biofilm formation assay is one of the most important methods of studying biofilm formation. In this study, the assay has been improvised to allow easy detection of biofilm formation on different substrata. The method has then been used to study growth conditions that affect biofilm formation, viz., the effect of pH, temperature, shaking conditions, and the carbon source provided. Glass, cellulose acetate, and carbon cloth materials were used as substrata to study biofilm development under the above conditions. The method was then extended to determine biofilm formation on the anodes of a microbial fuel cell in order to study the effect of biofilm formation on power production. A high correlation was observed between biofilm formation and power density (r = 0.951). When the electrode containing a biofilm was replaced with another electrode without biofilm, the average power density dropped from 59.55 to 5.76 mW/m2. This method offers an easy way to study the suitability of different materials to support biofilm formation. Growth conditions determining biofilm formation can be studied using this method. This method also offers a non-invasive way to determine biofilm formation on anodes of microbial fuel cells and preserves the anode for further studies.