Bacterial biofilms are dynamic and structurally complex communities, involving cell-to-cell interactions. In recent years, various environmental signals that induce the complex biofilm development of the Gram-positive bacterium Bacillus subtilis have been identified. These signalling molecules are often media components or molecules produced by the cells themselves, as well as those of other interacting species. The responses can also be due to depletion of certain molecules in the vicinity of the cells. Extracellular manganese (Mn2+) is essential for proper biofilm development of B. subtilis. Mn2+ is also a component of practically all laboratory biofilm-promoting media used for B. subtilis. Comparison of complex colony biofilms in the presence or absence of supplemented Mn2+ using microarray analyses revealed that genes involved in biofilm formation are indeed downregulated in the absence of Mn2+. In addition, Mn2+ also affects the transcription of several other genes involved in distinct differentiation pathways of various cellular processes. The effects of Mn2+ on other biofilm-related traits like motility, antimicrobial production, stress and sporulation were followed using fluorescent reporter strains. The global transcriptome and morphology studies highlight the importance of Mn2+ during biofilm development and provide an overview on the expressional changes in colony biofilms in B. subtilis.