Safety of potable reuse can be enhanced by improved water quality monitoring techniques for assessing water treatment processes. This study evaluated the efficacy of online bacterial counting for continuous monitoring of reverse osmosis (RO) membranes to remove bacteria using real-time bacteriological commercial counters and an on-site pilot-scale RO system. Prior to on-site assessments, the online bacterial counting was verified by comparing the measurement of fluorescent particles in water with flow cytometry. During a seven day pilot test of RO treatment at a water reclamation plant, online bacterial counts in RO permeate were monitored below 15 counts/mL; whereas the bacterial counts in RO feed water were approximately 2500 to 10,000 counts/mL. Removal rates of bacterial counts ranged from 2.6 to 3.1-log (average = 2.9-log) by continuously monitoring bacterial removal. This is greater than a 2-log reduction frequently determined using other water quality surrogates (i.e., electrical conductivity). Overall, the continuous monitoring of bacteria in RO feed and permeate can be implemented without the addition of chemicals to provide near real-time bacterial counts to measure their reduction after RO treatment. This can be developed for continuous performance monitoring of the RO process, providing greater assurance of microbial water quality after RO treatment. Display omitted •Efficacy of online bacterial counting for monitoring RO system integrity was assessed.•Real-time bacteriological commercial counters were used for this study's assessment.•Online bacterial counting was verified by measuring surrogates.•Bacterial counts in RO feed and permeate were monitored online at the pilot scale.•A range of 2.6 to 3.1-log reduction in bacteria was determined by online monitoring.