This study investigated ways of stimulating the biodegradation rates of the commonly applied herbicide, 3,4-dichloropropionanilide (propanil), and its metabolite, 3,4-dichloroaniline (DCA), as well as the growth rate of propanil- and DCA-degrading organisms in a mixed culture. Propionate, the other metabolite of propanil, stimulated the specific degradation rates of both propanil and DCA after a brief acclimation period. A metabolic model developed to characterise the metabolism of propanil and DCA biodegradation showed that the efficiency of oxidative phosphorylation (i.e. P/O ratio), which measures the metabolic efficiency, increased over time by 6- to 10-fold. This increase was accompanied by a 5- to 10-fold increase in the propanil and DCA biodegradation degradation rates. The biodegradation rates of the culture were unaffected when using an irrigation water matrix (Tejo river, Portugal), highlighting the utility of the culture for bioaugmentation purposes.