The present study aims to evaluate the nature and level of chemical pollution as well as the potential toxicity and ecotoxicity of an agricultural soil irrigated by the water of Litani River. Our findings showed that the soil was mainly contaminated by alkanes (hentriacontane, octadecane, hexadecane) and metal trace elements (nickel, vanadium, chromium, and manganese). Soil organic extracts showed high cytotoxicity against human hepatic (HepG2) and bronchial epithelial cells (Beas-2B). Soil ecotoxicity was revealed by seed germination inhibition of several plant species (wheat, clover, alfalfa, tall fescue, and ryegrass) ranging from 7 to 30% on the polluted soil compared to non-polluted one. In addition, significant decreases in telluric microbial biomasses (bacterial and fungal biomasses), quantified by phospholipid fatty acids (PLFA) analysis were observed in polluted soil compared to non-contaminated soils. The density of the arbuscular mycorrhizal fungal (AMF) spores isolated from the polluted soil was about 316 spores/100 g. Three main AMF species were identified as Funelliformis mosseae, Septoglomus constrictum, and Claroideoglomus lamellosum. Moreover, 16 indigenous plant species were inventoried with Silybum marianum L. as the dominant one. Plant biodiversity indices (Shannon, Simpson, Menhinick, and Margaleff) were lower than those found in other contaminated soils. Finally, it was found that all the present plant species on this polluted site were mycorrhized, suggesting a possible protection of these plants against encountered pollutants, and the possibility to use AMF-assisted phytoremediation to clean-up such a site.