The bioremediation of Antarctic soils is a challenge due to the harsh conditions found in this environment. To characterize better the effect of total petroleum hydrocarbon (TPH) concentrations on bacterial, archaeal and microeukaryotic communities in low (LC) and high (HC) hydrocarbon-contaminated soil samples from the Maritime Antarctic clone libraries (small-subunit rRNA genes) were constructed. The results showed that a high concentration of hydrocarbons resulted in a decrease in bacterial and eukaryotic diversity; however, no effect of the TPH concentration was observed for the archaeal community. The HC soil samples demonstrated a high relative abundance of bacterial operational taxonomic units (OTUs) affiliated with unclassified group TM7 and eukaryotic OTUs affiliated with unclassified fungi from Pezizomycotina subphyla. Chemical analyses of the LC and HC soil samples revealed the presence of negligible amounts of nitrogen, thereby justifying the use of biostimulation to remediate these Antarctic soils. Microcosm experiments showed that the application of fertilizers led to an increase of up to 27.8% in the TPH degradation values. The data presented here constitute the first step towards developing the best method to deploy bioremediation in Antarctic soils and provide information to indicate an appropriate action plan for immediate use in the case of new accidents.