The green transition and move towards safe and sustainable-by-design chemicals entail the need for new methods to study the biodegradability of UVCBs (substances of Unknown or Variable composition, Complex reaction products, and Biological materials). Standard simulation biodegradation tests have been developed for single substances and are generally not applicable for UVCBs. The aims of this study were (1) to combine a whole UVCB biodegradation test with a sensitive constituent-specific analytical technique, (2) to measure biotic and abiotic degradation of known and unknown UVCB constituents, and (3) to determine the impact of a wastewater treatment plant (WWTP) discharge on the constituent specific biodegradation in stream water. Lavender oil and black pepper oil are of significance in the perfume and cosmetics industries and served as model UVCBs. Stream water sampled upstream and downstream of a WWTP discharge point was characterized and used as inoculum (i.e., naturally and wastewater-adapted bacterial consortia). Tests were conducted in gastight headspace vials, and automated Arrow Solid Phase Microextraction GC-MS-scan was applied on unopened vials. Peak area ratios between biotic test systems and abiotic controls were used to determine primary biodegradation kinetics, and freshly spiked analytical references to separate biotic from abiotic degradation. Biodegradation half-times were below 20 days for all known (8–12) and unknown constituents (>78) in the essential oils. A dual-column GC-MS analysis produced a level 2 identification of 16 unknown lavender constituents. Biodegradation kinetics were similar in inoculum taken before and after the WWTP outlet, confirming that native stream microorganisms were competent degraders.