The cellular contents in bacteria are surrounded by an inner peptidoglycan cell wall in addition to an inner plasma membrane. Gram-negative bacteria also have an additional outer lipid bilayer. Specific antibacterials interfere with the synthesis of the cell wall, weakening the peptidoglycan scaffold within the bacterial wall, compromising the structural integrity. Since mammalian cells have a plasma membrane but lack the peptidoglycan wall structure, this class of antibacterials selectively targets the bacteria with no significant negative effect on the cells of the mammalian host.

Antibiotics that affect the structure of the cell wall act at different stages of peptidoglycan synthesis and cell wall construction. The precursor for the synthesis of peptidoglycan is a muramyl pentapeptide containing a terminal D-Ala-D-Ala. D-cycloserine inhibits two enzymes involved in the precursor synthesis, preventing both conversion of L-alanine to D-alanine by racemase, and the construction of D-alanyl-D-alanine by D-Ala-D-Ala ligase. In the cytoplasm, muramyl pentapeptide is anchored via a water-soluble UDP-glucosamine moiety. In the second phase of peptidoglycan construction, muramyl pentapeptide N-acetylglucosamine (muramyl pentapeptide anchored via a water-soluble UDP-glucosamine moiety) is transferred to a C55 undecaprenyl phosphate with the release of UMP to form a Lipid I intermediate. Tunicamycin inhibits the enzymatic conversion of the undecaprenyl phosphate to the lipid I intermediate, thus blocking the completion of peptidoglycan structure. An additional glycosylation step completes a peptidoglycan unit that is transported via its C55 lipid tail to the external periplasmic surface of the membrane. It is here that the peptidoglycan unit becomes integrated into the cell wall matrix. Bacitracin inhibits lipid phosphatase, preventing the release of the finished peptidoglycan from its C55 lipid carrier. Several transpeptidases and transglycosylases connect the newly formed peptidoglycan structures to the cell wall peptidoglycan matrix. ß-Lactam antibiotics (example Penicillin), with their structural similarity to the D-alanyl-D-alanine group within the peptidoglycan structure, compete for the binding sites of transpeptidases and prevent the assembly of the peptidoglycan layer in both Gram-positive and Gram-negative bacteria. Vancomycin, a glycopeptide antibiotic with a significantly larger structure than Penicillin, also prevents cell wall construction by interfering with transglycosylases. Its effectiveness is limited to Gram-positive bacteria because its large size prevents its penetration to the outer cytoplasmic membrane of Gram-negative bacteria.