Ready biodegradability tests (RBTs) are extensively used to screen the potential of chemicals to be biodegraded. The use of RBT protocols often results in large variations of test results that may lead to wrong interpretations. The present study aims to obtain a fundamental understanding of this variability. For this, we subjected the compounds 4-chloroaniline (4CA), carbamazepine (CBZ), metformin (MET), and N-methylpiperazine (NMP) to a variety of different test conditions. Inocula from five local wastewater treatment plants (WWTPs) were used in an attempt to enhance the Organisation for Economic Co-operation and Development (OECD) 310 biodegradability tests. The biodegradation capacity in RBTs, community composition and adaptation of the communities were compared after one week of pre-exposure in batch and four months exposure in chemostat. The results confirm that none of the test compounds is readily biodegradable in the standard OECD 310 RBT. However, when pre-exposure under either batch or chemostat conditions was included, 4CA was degraded in some cases and less variability among different inocula was observed for the transformation of MET. Bacterial communities from the five locations were found to be significantly different in composition from one another. In addition, pre-treatment performed before the RBT significantly changed the composition of each community. Results of this experiment show that short-term pre-exposure may increase the absolute number of degraders and deserves to be further investigated as a potential method to reduce the outcome variability of RBTs.