The structure of biofilms can be numerically quantified from microscopy images using structural parameters. These parameters are used in biofilm image analysis to compare biofilms, to monitor temporal variation in biofilm structure, to quantify the effects of antibiotics on biofilm structure and to determine the effects of environmental conditions on biofilm structure. It is often hypothesized that biofilms with similar structural parameter values will have similar structures; however, this hypothesis has never been tested. The main goal was to test the hypothesis that the commonly used structural parameters can characterize the differences or similarities between biofilm structures. To achieve this goal (1) biofilm image reconstruction was developed as a new tool for assessing structural parameters, (2) independent reconstructions using the same starting structural parameters were tested to see how they differed from each other, (3) the effect of the original image parameter values on reconstruction success was evaluated, and (4) the effect of the number and type of the parameters on reconstruction success was evaluated. It was found that two biofilms characterized by identical commonly used structural parameter values may look different, that the number and size of clusters in the original biofilm image affect image reconstruction success and that, in general, a small set of arbitrarily selected parameters may not reveal relevant differences between biofilm structures. Biotechnol. Bioeng. 2011; 108:1383–1394. © 2011 Wiley Periodicals, Inc.