Despite the use of levofloxacin prophylaxis during the neutropenic period after autologous peripheral blood stem cell transplantation, viridans group (VG) streptococcal bacteremia developed in 6 (16.2%) of 37 patients who underwent transplantation between 1 January and 25 February 2001 at the Mayo Clinic in Rochester, Minnesota. All 6 patients presented with fever and mucositis after a mean of 4.5 days of neutropenia, and 3 developed septic shock. All 6 VG streptococcal isolates from these patients exhibited distinct patterns on pulsed-field gel electrophoresis. All isolates had diminished susceptibility to levofloxacin, 5 to gatifloxacin, and 4 to moxifloxacin. Quinolone resistance was associated with mutations in the quinolone resistance-determining region of GyrA and (for 1 isolate) of ParC. The use of levofloxacin may select VG streptococci with diminished susceptibility to levofloxacin and other quinolones with enhanced activity against gram-positive organisms and, therefore, may not be optimal for preventing VG streptococcal bacteremia in neutropenic patients.