Streptococcus pneumoniae (S. pneumoniae) is a leading human pathogen, which takes large responsibility for severe otitis media, acute meningitis and septicaemia. It encodes up to three distinct sialidases: NanA, NanB and NanC, which are promising drug targets. Recent experimental studies have shown that these three sialidases might work together up to the ultimate step, where NanA and NanB produce N-acetylneuraminic acid (Neu5Ac) and 2,7-anhydro-Neu5Ac following the functions of sialidase and intramolecular trans-sialidase, whilst NanC carries on a ping-pong mechanism that produces or removes 2-deoxy-2,3-didehydro-Neu5AC. It is intriguing that these sialidases have similar active sites but operate via three distinct reaction pathways. To clarify this issue, herein we present the first systematic computational investigation on the catalytic pathways for S. pneumoniae NanA, NanB and NanC based on combined quantum mechanics/molecular mechanics simulations, and propose the most preferred routes for the three S. pneumoniae sialidases. Our findings support the mechanisms of NanA and NanC that were proposed by previous experimental studies, whereas the role of water in NanB was found to differ slightly from our current understandings. The mechanistic insights obtained from this work are expected to assist in the design of potent inhibitors targeting these key enzymes for therapeutic applications.