In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment.