Soil microbial communities play an important role in the biodegradation of different petroleum derivates, including hydrocarbons. Also other biological factors such as enzyme and respiration activities and microbial abundance are sensitive to contamination with petroleum derivates. The aim of this study was to evaluate the response of autochthonic microbial community and biological parameters (respiration, dehydrogenase and catalase activities, total microorganisms count) on contamination with car fuels and engine oils. The surface layer (0–20 cm) of Mollic Gleysol was used for the experiment. In laboratory conditions, soil was contaminated with the following petroleum substances: car fuels (petrol, diesel) and car engine oils (new and waste—after 10,000 km). The results demonstrated that, among the investigated hydrocarbon substances, petrol addition seemed to be the most toxic for the microbial activity of the investigated soil. The toxicity of the used hydrocarbon substances to microorganisms might be summarized as follows: diesel > new oil > waste oil > petrol. Species belonging to the genera Micrococcus and Rhodococcus were noted as the major autochthonic bacteria being present in soil contaminated with new automobile oil, whereas species of the genera Bacillus sp. and Paenibacillus sp. were identified in the combination treated with waste oil.