The biological effects induced by the pollutants present in soils, together with the chemical and physical characterizations, are good indicators to provide a general overview of their quality. However, the existence of studies where the toxicity associated to soils contaminated with mixtures of pollutants applying both in vitro and in vivo models are scarce. In this work, three soils (namely, Soil 001, Soil 002 and Soil 013) polluted with different concentrations of hydrocarbons and heavy metals were evaluated using different organisms representative of human (HepG2 human cell line) and environmental exposure (the yeast Saccharomyces cerevisiae, the Gram-negative bacterium Pseudomonas putida and, for the in vivo evaluation, the annelid Enchytraeus crypticus). In vitro assays showed that the soluble fraction of the Soil 001, which presented the highest levels of heavy metals, represented a great impact in the viability of the HepG2 cells and S. cerevisiae, while organic extracts from Soils 002 and 013 caused a slight decrease in the viability of HepG2 cells. In addition, in vivo experiments showed that Soils 001 and 013 affected the survival and the reproduction of E. crypticus. Altogether, these results provide a general overview of the potential hazards associated to three specific contaminated sites in a variety of organisms, showing how different concentrations of similar pollutants affect them, and highlights the relevance of testing both organic and soluble extracts when in vitro safety assays of soils are performed.