Stem cell tissue constructs are likely to come into contact with silver-based nanoparticles—such as silver chloride nanoparticles (AgCl–NPs)—used as microbicidals at the implant site or in cosmetics. However, the effect of silver-based nanoparticles on 3D cell cultures with potential for tissue engineering has received little attention. Here, we examined the effect of sub-lethal doses (5, 10 and 25 µg/mL, for 1, 7 and 21 days) of AgCl–NPs produced by ‘green’ bacterial-based synthesis on spheroid 3D cultures of human adipose tissue stem cells (ASCs). Light microscopy analysis revealed that the shape and diameter of ASC spheroids remained largely unchanged after AgCl–NP treatment. Flow cytometry analysis with 7-AAD and 2′,7′-dichlorofluorescein diacetate revealed no statistically significant differences in cell death but showed an increase of ROS levels for the untreated group and significant differences for the groups treated with 5 and 10 µg/mL at day 7 (p = 0.0395, p = 0.0266, respectively). Electron microscopy analysis showed limited cell damage in the periphery of AgCl–NP-treated spheroids. However, treatment with AgCl–NP had statistically significant effects on the secretion of IL-6, IL-8, IL-1β and IL-10 by spheroids, at specific treatment periods and concentrations, and particularly for IL-6, IL-8 and IL-1β. TGF-β1 and -β2 secretion also changed significantly throughout the treatment period. Our results indicate that, despite having little effect on cell viability and morphology, sub-lethal AgCL–NP doses modulate ROS production at day 7 for the groups treated with 5 and 10 µg/mL and also modulate the secretory profile of ASC spheroids. Thus, the use of skin implants or products containing Ag-NPs may promote long-term disturbances in subcutaneous adipose tissue homeostasis.