Significance Bacteria are protected from their surrounding environment by the peptidoglycan cell wall, which is a major target for antibiotics. Counterintuitively, cell wall assembly requires enzymes that cleave newly built peptidoglycan chains. Here, using nascent peptidoglycan we assembled in vitro, we characterized two membrane-bound glycosidases that are vital for proper cell division and elongation in Streptococcus pneumoniae . These enzymes were proposed to perform different chemical reactions. Instead, we show that they perform the same chemical reaction but cut the peptidoglycan backbone at different sites. We identify the mechanistic basis for cleavage site selection and also identify an amino acid switch that alters the cleavage chemistry. This work advances our understanding of how peptidoglycan glycosidases help build the cell wall.