Selective fluorogenic probes for the labelling of intracellular reduced glutathione (GSH), i.e. ortho-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA), have been encapsulated in polymeric nanoparticles (NPs) and the ability of the NPs to enhance uptake of the probe by microbial cells has been evaluated. Preparation of the probe-loaded NPs composed of Eudragit(®) E was based on an oil-in-water emulsification solvent evaporation method using an ultrasonic probe and polyvinyl alcohol as the surfactant. The encapsulation efficiency of the probes in lyophilized NPs was determined using high performance liquid chromatography (HPLC). A higher encapsulation rate of NDA than OPA was found: 47.6 ± 9.9 (n = 6) and 2.1 ± 0.2% (n = 3), respectively. The NDA-loaded particle diameter and zeta potential were 224.6 ± 14.7 nm and +40.9 ± 6.5 mV, respectively. After 20 min incubation of cultured Candida albicans yeast cells with either free NDA or NDA-loaded NPs (final NDA concentration 100 µM), cells were harvested and corresponding lysates were analysed using HPLC coupled with spectrofluorimetric detection. Incubation of cells with NDA-loaded NPs increased intracellular levels of NDA-GSH adduct by about nine-fold in comparison with the free probe. Adhesion on the cells and the penetration behaviour of NPs loaded with either NDA or fluorescent label (Nile Red) were characterized qualitatively by confocal laser scanning microscopy.