Elemental and isotopic composition of tunnel dusts collected from Jânio Quadros (JQ) and Maria Maluf (MM) tunnels in São Paulo city were analyzed aiming to determine the potential sources of trace elements in these environments. Sampling was performed in the summer and winter of 2017. Elemental mass fractions were determined by Instrumental Neutron Activation Analysis (INAA) and Graphite Furnace Electrothermal Atomic Absorption Spectrometry (GF-AAS) for 28 elements. Isotopic signatures of Pb and Zn were determined by Thermal Ionization Mass Spectrometry (TIMS) and Multicollector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS), respectively. The mean mass fractions of elements, such as Pb, Cd, Zn, Sb and Cu, were in general similar to the reported in the literature, in studies that demonstrated urban contamination by potentially toxic elements. Statistics demonstrated significant differences in mass fractions between the tunnels for most analyzed elements. Results showed that rare earth elements, U and Th, mostly associated with geogenic sources, presented higher concentrations in MM tunnels, while elements frequently related to vehicular emissions (Sb, Zn, Ba, Cu and Pb) presented mean mass fraction values higher in JQ tunnel, pointing to a more important contamination in JQ tunnel. No significant differences in the mass fractions between campaigns were observed, evidencing that tunnels are not much affected by external weather conditions. Pb isotopic analysis presented 206Pb/207Pb ratios between 1.1715 and 1.1791 and 208Pb/206Pb ratios between 2.0889 and 2.0961, which pointed out to a vehicular signature, related to tailpipe emissions. On the other hand, Zn isotope data also suggested a vehicular signature, but mostly related to the wear of tires and brakes. Grain size distribution analysis showed that the smallest fractions of dust contained, on average, about 10% of particulate matter of aerodynamic diameter smaller than 10 μm, fractions highly inhalable, what may be a concern to human health. Enrichment factors (EFs) showed that Cr, Zn, Cu, Cd, Pb and Sb are the most enriched elements, in both tunnels, but higher EFs were found for JQ tunnel. Principal Component Analysis revealed a profile for a crustal source, marked by U, Th and rare earth elements, whereas vehicular sources were characterized mostly by Pb, Ti and V from gasoline/diesel combustion; Pb and Ti from road paints; Sb, Ba and Zn from brakes, and Zn from wear of steel and tires. These results indicate an important anthropic impact in the tunnels and that vehicular traffic is the main source of potentially toxic elements.