After the Gulf War Oil Spill, there have been many investigations about distributions of oil-derived pollutants nearby areas, but lacking in ecotoxicological assessment. We evaluated the potential toxicity of asphalt mats, sediments, and biota (polychaetes, chitons, snapping shrimps, and crabs) by combining two bioassays (H4IIE-luc and Vibrio fischeri) and in situ microbial community (eDNA). Samples were collected from Abu Ali Island, and organic extracts were bioassayed and further fractionated according to the chemical polarity using silica gel column. Great aryl hydrocarbon receptor (AhR)-mediated potencies and inhibition of bioluminescence were mainly found in aromatics (F2) and saturates (F1) fractions of asphalt mat and sediments, respectively, while great toxicological responses in biota samples were found in resins and polar (F3) fraction. We also confirmed that potential toxicities of biota were species-specific; great AhR-mediated potencies were found in polychaetes and great bioluminescence inhibitions were found in crabs. In microbial communities, most genera (up to 90%) were associated with polycyclic aromatic hydrocarbons (PAHs)-degrading bacteria, supporting that PAHs are the primary stressors of the benthic community around Abu Ali Island. The present study provides useful information on the contamination status, risk assessment of environmental matrices and benthic organisms in Abu Ali Island.