Introduction of biofuels to the fuel matrix poses new questions and challenges. This study presents the behavior of microbial growth on physicochemical properties in different kinds of biodiesel/diesel mixtures that are produced from raw materials. A variety of fatty acid methyl ester samples were produced from raw materials such as sunflower oil, soybean oil, pomace oil, castor oil, palm oil and used frying oil. After these samples were produced they were mixed with conventional automotive diesel at concentrations of 10 and 20% v/v in respect with biodiesel. Also similar blends were prepared with the above produced methyl esters differentiating in which the antioxidant TBHQ had been added previously in concentration 0.1%. The outcomes indicated that in mixtures of methyl esters with an augmented content of saturated fatty acids such as palm methyl esters and monounsaturated fatty acids (i.e. pomace methyl esters) there was appearance of microbial growth in the fuel phase shown more quickly while the mixtures with TBHQ showed reduction in microbial load. Furthermore, in samples with a large augmentation in the number of acidity, the microbial load detected was negligible or zero. Samples with a zero or decreased microbial activity indicated lower pH values in the water phase.