Abstract Diesel fuel is a heavy fuel that composed of a complex mixture of hydrocarbons and used in combustion engines. Diesel fuel (DF) has lead to hazardous effect to the environment and can cause some specific harmful effects in human health such as irritation to eye, skin and respiratory system and some carcinogenic effects. It is a common pollutant to marine and soil environment. In the present work, isolation of filamentous fungi capable of diesel fuel biodegradation from the industrial region in south Jeddah, Saudi Arabia was conducted. The isolated fungi were Aspergillus flavus, A. fumigatus, A. niger, A. ustus, Alternaria alternata, Penicillium corylophilum, P. fellutanum and Rhizopus rhizodopodiformis. A. ustus was the most efficient fungus for degradation of 1% of DF and A.alternata was less efficient. Time course biodegradation of DF by A. ustus and A.alternata indicated that 7 days of fermentation was satisfactory to biodegrade 0.5ml DF. The efficiency of A. ustus and A.alternata to biodegrade different levels of DF (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml/50ml medium) indicated that as the DF concentration increased the degradation activity decreased by about 25% for A. ustus and about 22.9% for A.alternate, which proved that A. alternata is more efficient in degrading of higher DF than do A. ustus. Mixed culture of A. ustus and A.alternata was applied in different inoculum levelsin the ratio 1:1 (1.6, 2.4 and 3.2 X 106 spores/ml), indicated that the biodegradation was considerably enhanced upon using mixed culture, as the inoculum size increased from 1.6, 2.4 and 3.2 X 106 spores/ml the biodegradation activity increased noticeably. Statistical design of two phase multifactorial optimization (Plackett-Burman and Box-Behnken) were carried out to optimize cultural conditions to increase the efficiency of mixed culture for biodegradation of 4ml DF. The data indicate that factors in trail (4) was the best for complete biodegradation of 4ml DF/25ml basal medium after 7 days (increased the biodegradation rate by about 2%). The data of Plackett-Burman design also indicated that the most three significant factors on DF biodegradation were CaCl2, FeCl3 and (NH4)2 HPO4. These three factors were further investigated using a response surface methodology of Box-Behnken. The results of Plackett-Burman and Box-Behnken statistical designs indicated that the optimized conditions accelerated the rate of DF biodegradation at 7 days of fermentation and assessed the validity of the model. In an applied trial, hydrocarbon free soil contaminated with 5ml DF was completely became diesel fuel free when amended with the fungal consortium under the best optimized conditions.