This study systematically analyzes the dark shale samples of the Silurian Qusaiba Formation from the Tayma Quadrangle outcrop section in the northwest of Saudi Arabia, and assesses the source and nature of its organic matter and the main sedimentary environmental conditions during accumulation of organic matter. The Qusaiba shale samples are characterized by total organic carbon (TOC) and sulfur contents with total values in the range of 0.87–1.76 wt. % and 0.59–4.64 wt. %, respectively, indicating a marine setting ranging from dysoxic to anoxic environmental conditions. The biomarkers are characterized by a relatively low Pr/Ph ratio between 0.50 and 1.24. The abundance of tricyclic terpanes and high C27 and C29 regular steranes equated to C28 regular sterane, providing evidence that the organic matter derived primarily from marine organisms, including algal and other aquatic organic matter and some terrigenous land plants, and was deposited under dysoxic to anoxic environmental conditions. The significant low oxygen environmental conditions may contribute to preservation of organic matter during deposition. The inorganic geochemical indications suggest that the Qusaiba shales were accumulated in a warm–humid climate and with low salinity stratification conditions of the water columns.