Near-infrared (NIR) fluorophores show superior in vivo imaging properties than visible-light fluorophores because of the increased light penetration in tissue and lower autofluorescence of these wavelengths. We have recently reported that new NIR cyanine dyes containing a novel C4'-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4'-O-aryl variants. In this study, we synthesized two NIR cyanine dyes with the same core structure and charge but different indolenine substituents: FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, and FNIR-G-765 bearing a combination of two sulfonates and two guanidines, resulting in zwitterionic charge with distinct cationic moieties. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-Z-759 and FNIR-G-765 with panitumumab (pan) at antibody-to-dye ratios of 1 : 2 or 1 : 5. One-to-five conjugation of pan-to-FNIR-G-765 was not successful due to aggregate formation during the conjugation reaction. Conjugates of both dyes to pan (2 : 1) demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower accumulation in the mouse liver, resulting in higher tumor-to-liver ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-G-765 conjugates, especially in the abdominal region. Moreover, from a chemistry point of view, mAb conjugation with FNIR-Z-759 has an advantage over FNIR-G-765, because it does not form aggregates at high dye-to-mAb ratio. These results suggest that zwitterionic cyanine dyes are a superior class of fluorophores for conjugating with mAbs for fluorescence imaging applications due to improving target-to-background contrast in vivo. However, zwitterionic cyanine dyes should be designed carefully, as small changes to the structure can alter in vivo pharmacokinetics of mAb-dye conjugates.