<p>This study explored the synergistic effects of soybean oligosaccharides (SBOS) and <i>Chlorella pyrenoidosa</i> (C<i>. pyrenoidosa</i>) on water quality and microbial community structure of aquaculture effluent in a biofloc system. Five treatments (5 replicates, C/N = 15) were tested for 9&#xa0;days: CON group (control, no treatment), GLU group (glucose), SBOS group (2.5% SBOS and 97.5% glucose), CP group (glucose and C<i>. pyrenoidosa</i>), and CS group (2.5% SBOS, 97.5% glucose and C<i>. pyrenoidosa</i>). C<i>. pyrenoidosa</i> concentration was 5.37 × 10⁵ cells/mL. On day 1, nitrate nitrogen and total nitrogen in CP and CS groups were significantly lower than GLU group; on day 5, ammonia nitrogen content in CS group was significantly lower than others experimental groups; on day 7, nitrite nitrogen content in CP and CS groups was significantly lower than others experimental groups (<i>P</i> &lt; 0.05); on day 3 biofloc volume (FV) in CS group showed a significant increase compared to CON and GLU groups (<i>P</i> &lt; 0.05). All experimental groups reduced turbidity compared to CON group (<i>P</i> &lt; 0.05). SBOS and C. <i>pyrenoidosa</i> demonstrated significant synergistic effects on water quality and biofloc formation: nitrite reduction (day 1), increased FV (day 3), and nitrate reduction (days 5 and 7) (<i>P</i> &lt; 0.05). Compared with other experimental groups, the abundance of <i>Aeromonadaceae</i> decreased significantly, and <i>Bacillus</i> and <i>Mycobacteria</i> increased significantly in CS group, enhancing nitrogen cycling and organic matter degradation pathways (<i>P</i> &lt; 0.05). These findings suggest that SBOS and C<i>. pyrenoidosa</i> exhibit synergistic effects in treatment of effluent in biofloc system, and efficiently remove nitrogen and optimize microbial community structure.</p>