Herein, we synthesized some ultra small silver nanoclusters (AgNCs) and palladium nanoclusters (PdNCs) protected by L-glutathione (L-GSH) reduced ligand to study their antimicrobial properties in the visible and dark conditions. These clusters can be dissolved, dried, and redissolved in water without suffering from any agglomeration or degradation. The particles sizes of the synthesized nanoclusters were around 1 nm, which investigated by high-resolution transmission electron microscopy (HRTEM). The optical properties of the synthesized nanoclusters were studied by UV–vis spectroscopy. Agn(SG)m clusters exhibited two plasmon peaks in the visible region at 478 and 641 nm. However, Pdn(SG)m clusters showed a steep rise absorption in the short wavelength range of the spectrum and featureless in the visible region. Fourier transform infrared spectroscopy (FTIR) was measured for the free ligand and protected clusters Agn(SG)m and Pdn(SG)m. Compared with palladium nanoclusters (in vitro), silver nanoclusters exhibited higher biocidal activity (MIC). The influence of silver and palladium nanoclusters (0.0, 2.5, 3.5, 5.0, 8.0 mg/l) on soil fungal community density was investigated under dark and light condition. Interestingly, silver NPs were more effective than palladium nanoclusters. Moreover, silver NPs were more effective in light than in the dark. However, palladium nanoclusters showed more or less similar effect under both contrasting conditions. This might be attributed to the disappearance of the plasmonic effect of palladium clusters in the UV–vis region. The novelty of our method is due to using ligand (L-GSH) as a capping agent with palladium. Besides, L-glutathione is considered as a part of the microbial system to detoxify metals. This guarantees the safety of the synthesized nanoparticles to be applied commercially than the naked isotopes. These nanoclusters could be considered a basic unit for medicine industry such as drug delivery tools or applied in paints industry (antimicrobial agents).