Silver nanoparticles (AgNPs) are widely incorporated into different hygiene, personal care, and healthcare products. However, few studies have been undertaken to determine the effects of biogenic AgNPs on human health. The effect of biosynthesized AgNPs using the fungus Aspergillus tubingensis culture was evaluated on human umbilical vein endothelial cells (HUVECs), normal human fibroblasts (FN1), human hepatoma cells (HEPG2) and a Galleria mellonella model. HUVECs were more susceptible to biogenic AgNPs than normal fibroblasts FN1 and intense cytotoxicity was observed only for very high concentrations at and above 2.5 μM for both cells. Normal human fibroblasts FN1 exposed to AgNPs for 24 h showed viability of 98.83 ± 8.40% and 94.86 ± 5.50% for 1.25 and 2.5 μM, respectively. At 5 and 10 μM, related to the control, an increase in cell viability was observed being 112.66 ± 9.94% and 117.86 ± 8.86%, respectively. Similar results were obtained for treatment for 48 and 72 h. At 1.25, 2.5, 5 and 10 μM of AgNPs, at 24 h, HUVECs showed 51.34 ± 7.47%, 27.01 ± 5.77%, 26.00 ± 3.03% and 27.64 ± 5.85% of viability, respectively. No alteration in cell distribution among different cycle phases was observed after HUVEC and normal fibroblast FN1 exposure to AgNPs from 0.01 to 1 μM for 24, 48 and 72 h. Based on the clonogenic assay, nanoparticles successfully inhibited HEPG2 cell proliferation when exposed to concentrations up to 1 μM. In addition to that, AgNPs did not induce senescence and no morphological alteration was observed by scanning electron microscopy on the endothelial cells. In the larvae of the wax moth, Galleria mellonella, a model for toxicity, AgNPs showed no significant effects, which corroborates to the safety of their use in mammalian cells. These results demonstrate that the use of A. tubingensis AgNPs is a promising biotechnological approach and these AgNPs can be applied in several biomedical situations.