Synthetic amorphous silica nanoparticles (SAS NPs) have been used in various industries, such as plastics, glass, paints, electronics, synthetic rubber, in pharmaceutical drug tablets, and a as food additive in many processed foods. There are few studies in the literature on NPs using gene mutation approaches in mammalian cells, which represents an important gap for genotoxic risk estimations. To fill this gap, the mouse lymphoma L5178Y/ assay (MLA) was used to evaluate the mutagenic effect for five different concentrations (from 0.01 to 150 μg/mL) of two different sizes of SAS NPs (7.172 and 7.652 nm) and a fine collodial form of silicon dioxide (SiO). This assay detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The results obtained indicate that the two selected SAS NPs are mutagenic in the MLA assay, showing a concentration-dependent effect. The relative mutagenic potencies according to the induced mutant frequency (IMF) are as follows: SAS NPs (7.172 nm) (IMF = 705.5 × 10), SAS NPs (7.652 nm) (IMF = 575.5 × 10), and SiO (IMF = 57.5 × 10). These results, obtained from mouse lymphoma cells, support the genotoxic potential of NPs as well as focus the discussion of the benefits/risks associated with their use in different areas.