<p>While rotifers are important first live feed in marine fish hatcheries, maintaining the rotifer culture collection by feeding live microalgae and water exchange is laborious and costly. It is therefore necessary to streamline the culture collection protocol and reduce operating costs. The use of microalgal paste in rotifer production is well documented, but there is limited information on its use in rotifer culture collection because of its ability to deteriorate water quality, such as increasing unionized ammonia (NH₃). This study aimed to determine the effectiveness of bioflocs (BF) in regulating NH₃ in rotifer culture collection fed microalgal paste Fresh Chlorella V12 (CV12) under zero-water exchange (ZWE) conditions for 42&#xa0;days. The marine rotifers <i>Brachionus plicatilis</i> were subjected to six treatments, namely: (1) <i>Nannochloropsis oculata</i> (Nanno) × weekly water exchange (WWE), Nanno × WWE as the control, (2) CV12 × WWE, (3) Nanno × ZWE, (4) CV12 × ZWE, (5) Nanno + BF × ZWE, and (6) CV12 + BF × ZWE. Rotifer density was estimated every 3&#xa0;days, while NH₃ was measured weekly. Bacterial compositions were determined. On day 42, the lowest NH₃ was observed in CV12 + BF × ZWE, which was significantly different from CV12 × WWE and CV12 × ZWE. Rotifer density was higher in Nanno + BF × ZWE and CV12 + BF × ZWE than in CV12 × ZWE, but egg-bearing rotifers were similar in all treatments on day 42. Single initial inoculation of BF maintains water quality by regulating NH₃ concentration via the activity of ammonia oxidizer <i>Nitrosomonas nitrosa</i> and the bacterial groups under phylum Chloroflexota in the long-term <i>B. plicatilis</i> culture collection and that microalgal paste combined with BF can be a cost-effective alternative to live microalgae.</p>