In cork industry, the operation of boiling raw cork generates large volumes of wastewater named Cork Boiling Wastewater (CBW). The main characteristics are the low biodegradability and medium to low acute toxicity, resulting in the necessity of designing advanced biological treatments by possible conventional activated sludge adaptation. In order to evaluate the variation of bacterial population along that process, a study based on optical microscopy, plate count, DNA extraction, qPCR and massive sequencing techniques was performed. Results showed a diminution of the total and volatile solids (TSS and VSS), jointly with a decrease in DNA concentration, general bacteria (16 S) and ammonia-oxidizing bacteria (AOB). After a few hours of testing, diverse microbiological species died while others showed a possible adaptation of the biological system, accompained by a dissolved organic carbon (DOC) reduction. In addition, toxicity tests based on activated sludge showed the development of chronic toxicity through the contact time. Combination of classical and advanced microbiological techniques, such as quantitative real time Polymerase Chain Reaction (qPCR) and metagenomics, was essential to predict the variation of species during the experiment and to conclude if effective biological adaptation could be finally attained for the target complex wastewater.