Four new Ascomycete fungi capable of degrading diesel oil were isolated from sediments of a river estuary mainly contaminated by shipyard fuels or diesel oil. The isolates were identified as species of Lambertella, Penicillium, Clonostachys, and Mucor. The fungal candidates degraded and adsorbed the diesel oil in suspension cultures. The Lambertella sp. isolate displayed the highest percentages of oxidation of diesel oil and was characterised by the capacity to utilise the latter as a sole carbon source. This isolate showed extracellular laccase and Mn-peroxidase activities in the presence of diesel oil. It was tested for capacity to accelerate the process of decontamination of total petroleum hydrocarbon contaminated sediments, co-composted with lignocellulosic residues and was able to promote the degradation of 47.6% of the TPH contamination (54,074 ± 321 mg TPH/Kg of sediment) after two months of incubation. The response of the bacterial community during the degradation process was analysed by 16S rRNA gene meta-barcoding.