Most biodegradation tests are conducted using single chemicals at high concentrations, although these chemicals are present in the environment as mixtures at low concentrations. A partitioning-based platform was recently developed for biodegradation testing of composed mixtures of hydrophobic chemicals at ng/L to μg/L concentrations. We used this platform to study the concentration and mixture effect on biodegradation kinetics. Biodegradation tests were conducted in 20 mL vials using environmental water samples as inocula. Passive dosing was applied (1) to vary initial test concentrations of individual test compounds and (2) to vary the number of mixture components between 1 and 16. Automated solid-phase microextraction coupled to gas chromatography-mass spectrometry was used to measure substrate depletion relative to abiotic controls. The number of mixture components had no or only a limited effect on the biodegradation half times for three compounds when tested at environmentally relevant concentrations. In contrast, longer lag phases and half lives were observed for single compounds when tested at higher concentrations that approached aqueous solubility. The obtained results support that simultaneous testing of multiple chemicals at low concentrations can accelerate the generation of biodegradation kinetic data, which are more environmentally relevant compared with data from tests conducted with single chemicals at much higher concentrations.