The aim of this research was to investigate the effect of new, non-conventional starter culture on the kinetics of the lactose transformation during milk fermentation by kombucha, at pH 5.8; 5.4; 5.1; 4.8; and 4.6, at two different temperatures 37 °C and 42 °C. Milk fermentation at 42 °C lasted significantly shorter (about 5 h, 30 min) compared to the fermentation at 37 °C. Changes of lactose concentration at the both temperatures are consisting of two retaining stages and very steep decline in-between. The analysis of the rate curves showed that the reaction rate passes through the maximum after 9 h, 30 min at 37 °C and after 4 h at 42 °C. The sigmoidal saturation curve indicates a complex kinetics of lactose fermentation by kombucha starter.