Ultrasonic Spray Pyrolysis (USP) possesses a great potential for production of higher quantities of gold nanoparticles (AuNPs), thus overcoming the problem of batch-to-batch variations in their properties. Recently, we demonstrated that USP with an additional evaporation chamber (modular USP) led to a better size control of AuNPs. However, their morphology, stability, toxicity, and immunomodulatory properties have not been investigated completely. Here, two types of spherical AuNPs were produced by using different USP parameters, followed by their stabilization in Na-citrate solution. No significant changes in their size, agglomeration, andz-potential occurred 3 months after their initial production in citrate solution. However, the conditioning of AuNPs in serum-containing cell culture media for 24 h induced an increase in the AuNPs’ hydrodynamic size and a red shift in their Surface Plasmon Resonance, pointing to their instability in biological media. Cytocompatibility tests showed that the produced AuNPs were internalized by L929 cells and primary human monocytes and were not cytotoxic at the concentrations lower than 200 μg/mL, but they exhibited antiproliferative and anti-inflammatory effects, respectively. AuNPs reduced the percentage of CD14+CD16+but not CD14lowCD16+monocytes in vitro and reduced the expression of CD86, HLA-DR, TNF-α, and IL-12/IL-23 by these cells. These results indicate that the anti-inflammatory effects of citrate-capped AuNPs produced by modular USP could be beneficial for their application in the treatment of inflammatory conditions.