In laboratory conditions the influence of Diesel fuel oil on the total number bacteria, fungi, actinomycetes and content of microbial biomass in soil was studied. The fuel oil was applied at dosage of 1, 2 and 5 g/100 g of soil. Samples of soil were analysed 1, 7, 14, 21 and 150 days after oil applied. Microbiological analyses involved determinations of the total number of bacteria and actinomycetes (on Bunta-Roviry medium) and fungi (on Martin's agar) by the plate method. The content of microbial biomass in soil was determined using the physiological method of Anderson and Domsch. Diesel fuel oil application caused the changes of total number of investigated groups of soil microorganisms. These results were confirmed by the investigated groups of soil microorganisms. These results were confirmed by the investigation on microbial biomass in soil. In the most of cases the fuel oil was the most stimulated for the number of bacteria and the most inhibited for actinomycetes in comparison with soil without oil (control). From soil with Diesel fuel oil addition Pseudomonas, Arhtrobacter, Bacillus and Penicillium, Aspergillus, Fusarium, Trichoderma strains were isolated. The possibility of the fuel oil as source of carbon utilization by these strains will be investigate.