We developed fluorine-18 ([(18)F]) labeled methyl 6-deoxy-6-fluoro-4-thio-α-d-maltotrioside ([(18)F]MFTMT) for bacterial imaging and evaluated its stability and efficacy in vitro and in vivo. We found that Staphylococcus aureus (S. aureus) internalized [(18)F]MFTMT whereas Escherichia coli (E. coli) and CHO-K1 cells did not, in in vitro. Positron emission tomography imaging with [(18)F]MFTMT revealed that radioactivity accumulated not only in the S. aureus-infected group but also in the E. coli-infected and non-infectious inflammation groups. Further studies revealed that rat serum digested [(18)F]MFTMT into [(18)F]-methyl 6-deoxy-6-fluoro-4-thio-α-d-maltoside ([(18)F]MFTM), while [(18)F]MFTMT was stable in human serum for 210 min. [(18)F]MFTM was identified as the only radioactive metabolite in vivo. Similar to [(18)F]MFTMT, [(18)F]MFTM was internalized only by S. aureus. [(18)F]MFTM was identified as the only radioactive metabolite in vivo. We found that the sodium-glucose co-transporter 1 (SGLT1) is expressed in inflammatory tissue, and SGLT1 overexpressing cells showed increased retention of [(18)F]MFTMT and [(18)F]MFTM in vitro. Our study showed that the thio-glycosyl bond is stable against enzymatic digestion, and maltotetraose or a longer maltodextrin backbone is desirable for bacteria-specific imaging to avoid nonspecific uptake by SGLT1.