The pathogenic opportunist and causative agent of the endemic disease cholera Vibrio cholerae naturally occurs in two distinct forms, in a single cell state and in so-called biofilms, consisting of cells in a polysaccharide matrix. It was observed that during biofilm development an initially geno- and phenotypically homogenous population of V. cholerae cells diversifies and adapts to the biofilm environment, leading to a higher phenotypic diversity. This study investigates if the phenotypic diversity is due to genotypic differences, by focusing on one aspect of mutagenesis, the spontaneous mutation of cells. Therefore, mutation frequencies were determined using the lacI repressor as a mutation trap. The genetically-engineered construct, transformed into the bacterias chromosome via natural transformation upon induction of natural competence by growth on chitin, enabled the examination of evolutionary processes in real-time on a molecular level. The mutated cells thereby arising, were localized in the biofilm by confocal laser scanning microscopy. The mutation frequency of harvested biofilm cells was established and compared to the frequency determined for planktonic cells. In a parallel approach, mutation frequencies were estimated by measuring the spontaneous acquisition of antibiotic resistance to streptomycin, spectinomycin or nalidixic acid. The study indicates that the mutation frequencies of both cell states are within the same order of magnitude, i.e. approximately one in a million cells mutates. However, the results imply that, while posing several challenges, a determination of the mutation rate instead of the mutation frequency would be more conclusive.