Biological treatment is gaining ground as a means to enhance removal of total organic carbon (TOC) as part of a multi‐barrier treatment train for water reuse. Here we applied biodegradable dissolved organic carbon (BDOC) analysis to evaluate the extent of removal of various TOC fractions through a pilot‐scale water reuse train employing flocculation/sedimentation, ozone, biologically active carbon (BAC), and granular activated carbon (GAC). BDOC analysis highlighted GAC and ozone treatments as critical to non‐biodegradable dissolved organic carbon removal and the need to optimize BAC performance to maximize GAC adsorption capacity. BDOC analysis was further applied to benchmark process performance to operational conditions, such as empty bed contact time (EBCT), occurrence of nitrification, and operational upsets. The lower EBCT proved to be less resilient to nonideal conditions. Overall, BDOC analysis proved an asset for understanding and improving operation of ozone/BAC/GAC treatments for water reuse.