Kuwait’s only fresh water source was contaminated with crude oil in the First Gulf War (1991), where oils wells were destroyed, and seawater was used to extinguish oil fires. Limited research has focused on the effect of the contamination and microbial communities along the down gradient groundwater flow path. Here, the bacterial, archaeal and eukaryotic communities at 15 sites along the contaminated groundwater flow path were investigated. Characteristics of the hydrocarbon contaminants present along the transect were also examined for the first time. This study detected potential hydrocarbon degrading microorganisms such as Hyphomicrobiaceae, Porphyromonadaceae and Eurotiomycetes, and a significant correlation of the microbial community structures with total petroleum hydrocarbon (TPH) concentration and electric conductivity (EC) measurements. Anoxic conditions appear to be dominating the sites closer to the contamination source, with methanotrophs and anaerobic organisms present. The TPH consisted of a prominent unresolved complex mixture (UCM), indicating the same persistent characteristics as identified in previous research. Lower concentrations further along the groundwater flow path probably due to dilution as indicated by correlations of TPH and EC, and contaminant characteristics. The findings contribute to understanding the groundwater pollution in Kuwait, and give further insight into designing potential future remediation efforts.