Zebrafish embryotoxicity and dioxin-like activity levels were tested for particulate air samples from an urban background site in Barcelona (Spain). Samples were collected during 14 months, and maximal values for both biological activities corresponded to samples collected during late autumn months, correlating with elevated PAH levels. Vehicle and combustion emissions appeared as the potentially most toxic sources, whereas total PM mass and mineral content appeared to be poor predictors of the biological activity of the samples. Samples simultaneously collected at different particle size cut-offs (10, 2.5, and 1 μm) did not differ significantly in dioxin-like PAH levels and biological activity, indicating that the sub-micron particle fraction (PM1) concentrated essentially all observed toxicity. Our results support the need for a tighter control on sub-micron particle emissions and show that total PM mass and, particularly, PM10, may not fully characterize the toxic potential of air samples.