<p>Many people spend time indoors. However, the chemical characteristics and toxicity of settled dust still need to be examined since limited data on their key parameters are available. Therefore, the aim of this study was to investigate the exposure risks of settled house dust and office dust using multiple parameters. Moreover, the risks of indoor dust from metals and polymers were modelled. Furthermore, the human health risk was examined via in vitro human lung epithelial cells. The results from the chemical analysis revealed that oxidative indicators were greater in settled house dust than in office dust. For example, higher O, N, O/C, S=O, and C-O levels were detected in house dust than in office dust. Oxidative potential of house dust was significantly (1.07 ± 0.15) higher than in office dust (0.97 ± 0.09). Moreover, office dust was characterized mainly by polyethylene terephthalate, whereas house dust was characterized mainly by polyamide. The results of the analysis of potentially toxic metals (As, Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) by ICP–MS indicated that the concentrations of metals, with the exceptions of Zn, were greater in office dust than in house dust. The cytotoxic responses of lung cells to mitochondria indicated greater inhibition of these cells to house dust (22.4 ± 15.7–23.9 ± 15.3% inhibition) than to office dust (12.0 ± 14.1–19.6 ± 14.2% inhibition, except low dose). Similar results were observed for the lipid membranes and antioxidants. Overall, the results showed that differences in chemical indicators had the determining role on the cytotoxic responses.</p>