A vital goal of sustainable aquaculture is understanding the microbial community assembly and dynamics under different settings, thus providing insights for more holistic management and manipulation. Regarding this, we explored the microbial communities in an Integrated Multi-Trophic Aquaculture (IMTA) system composed of sea urchins (Paracentrotus lividus), mullet fish (Mugil cephalus), and green seaweed (Ulva fasciata) as a biofilter. Over twelve weeks, the system was monitored for water quality parameters, and samples were taken for 16S rRNA amplicon sequencing to characterize and determine the influence of water quality parameters on the microbial community in the system. The study highlights the essential role of microbial communities in sustaining the ecological balance and functionality of IMTA systems. Water samples exhibited higher microbial diversity, driven by dynamic environmental conditions, while host-associated microbiomes showed niche specialization, reflecting their roles in nutrient assimilation, immune support, and detoxification. Ulva fasciata demonstrated its effectiveness as a biofilter, maintaining stable nutrient loads and microbial composition. The presence of beneficial microbes, such as Pseudoalteromonas, Rubidimonas, Granulosicoccus, Nonlabens, and Alteromonas, and the lower abundance of Vibrio spp. underscore the potential of Ulva in pathogen control and nutrient cycling. Nutrient availability, particularly nitrogen and phosphorus, influenced microbial interactions, which shaped community structure. Temporal shifts in microbial composition due to water quality changes were buffered by the system’s resilience, particularly through the stabilizing effects of Ulva. These findings reinforce the importance of microbial diversity and interactions in improving sustainability and productivity in IMTA systems. The study provides a foundation for understanding the microbial community in IMTA in enhancing nutrient recycling, pathogen control (using Ulva as a biofilter), and system stability, paving the way for more sustainable aquaculture practices.