The effect of operating conditions during molinate degradation by the defined mixed bacterial culture DC, previously described as able to mineralize molinate, was evaluated in a batch reactor. Parameters such as the rate of molinate degradation, the dissolved organic carbon (DOC) consumption and the accumulation of molinate degradation products were monitored along the culture growth. The effect of conditions such as temperature, pH, aeration rate, salinity, and presence of additional carbon and/or nitrogen sources, was tested independently. Degradation of molinate in river water was also evaluated. Culture DC was able to grow and to mineralize molinate at all the conditions assayed. Temperature was the factor with the strongest influence on bacterial growth and molinate mineralization. The lowest and the highest rate values of growth (0.010 and 0.110 h −1) and of molinate degradation (0.027 and 0.180 g molinate g −1 cell dry wt h −1) were obtained at 15 and 35 °C, respectively. In cultures with approximately 187 mg l −1 of molinate, 2-oxo-molinate was the major molinate degradation product accumulated in the medium, in concentrations below 0.133 mg l −1. Degradation of molinate was also evaluated in a continuous stirred tank reactor (CSTR). Operating the CSTR at a hydraulic retention time (HRT) of 83 h, fed with medium containing molinate concentrations ranging from 1 to 3 mM, culture DC degraded the herbicide with specific degradation rates similar to those obtained in the batch systems.