Shatt Al-Arab River in Basrah, Iraq represents an important source of fresh water so the current study aimed to determine the extent of pollution of the Shatt al-Arab River with some heavy metals and hydrocarbons by measuring their concentrations in sediment samples collected from different stations along the river, as well as the adoption of biomarkers represented by bacteria and fungi in determining the extents of such pollution. The results indicate that the river is contaminated with the following metals:: Ni, Cr, Zn, Pb, Cu and Cd. The total mean concentrations of heavy metals in the exchangeable part were high with 55.23, 53.76, 40.52, 33.84, 32.70 and 14.88 µg/g, respectively. The results also indicated a high concentration of hydrocarbons where the total mean was 28.52 µg/g. Bacteria and fungi represent vital indicators of heavy metals and hydrocarbons pollution in the environment. Different types of bacteria (Sphingomonas paucimobilis, Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus thoraltensis, Staphylococcus lentus, Leuconostoc cremoris and Leuconostoc mesenteroides) and fungi (Aspergillus terreus, Aspergillus fumigatus, Aspergillus niger, Curvularia sp., Eurotium cristatus, Talaromyces flavus, Mucor sp., Rhizopus sp., Trichoderma sp., Dichotomomyces cejpii, and Penicillium spp.) have been isolated from the same sediment. Isolated of specific genera from bacteria and fungi like these isolated in the present study from the sediment of all studied stations reinforces the rest of the results recorded during the current study and confirms the pollution of the river with the studied pollutants.