Chloramphenicol-loaded (CHL) poly-d,l-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) were prepared by emulsification solvent evaporation technique either by using polyvinyl alcohol (PVA) as emulsion stabilizer or polysorbate-80 (PS-80) as surfactant and characterised by transmission electron microscopy, zeta-potential measurements. The NPs were radiolabeled with technetium-99m ((99m)Tc) by stannous reduction method. Labeling conditions were optimised to achieve high-labeling efficiency, in vitro and in vivo (serum) stability. The labeled complexes also showed very low transchelation as determined by DTPA challenge test. Biodistribution studies of (99m)Tc-labeled complexes were performed after intravenous administration in mice. The CHL-loaded PLGA NPs coated with PS-80 exhibited relatively high brain uptake with comparatively low accumulation in bone marrow to that of free drug and CHL-loaded PLGA NPs (PVA, used as emulsion stabilizer) at 24 h post injection time period. This indicates the usefulness of the above delivery system for prolonged use of the antibiotic.