Abstract: Sustainable aquaculture needs to be considered when it comes to the utilization of water resources. The aim of this study was to apply biomonitoring using macrobenthic structures on both spatial and temporal applications of monoculture and integrated multi-trophic aquaculture (IMTA) at Tembelas Island, Kepulauan Riau Province, Indonesia. Samples of sediment were taken from three fish farm locations, namely from an IMTA site, a monoculture site, and a reference site. Macrobenthic organisms obtained through rinsing, sieving, and sorting were then identified under a stereo-microscope. Diversity of the macrobenthic assemblages was analyzed with a Shannon-Wiener index (H’). Equitability was expressed through Pielou’s evenness index. Finally, Bray-Curtis’ non-metric multi-dimensional scaling (NMDS) was used for similarities derived from log (X+1) transformed macrobenthic abundance to provide a visual representation of differences in their structure between sites over time. Results showed polychaetes exhibited differences in both variation and abundance of genera between the farm and reference site. The assemblage of macrobenthos at the IMTA site consisted of 9 genera of gastropods, 3 genera of bivalves, 5 genera of polychaetes, and 2 genera of crustaceans. At the monoculture site, 12 genera of gastropods, 4 genera of bivalves, 8 genera of polychaetes, 1 genera of crustaceans, and 1 genera of ophiuroid were observed. A relatively high abundance was observed at the reference site, with 27 genera of gastropods, 11 genera of bivalves, 3 genera of polychaetes, and 1 genera of crustacean. The favorable water conditions and possible absence of waste input from aquaculture resulting in a more suitable habitat for macrobenthic life may explain this relative abundance. Some of them were recognised as opportunistic taxa, i.e., Capitella sp., Heteromastus sp., and Lumbrinereis sp. Based on the diversity and evenness indices and the MNDS ordination, it can be concluded that the application of IMTA systems results in a suppressed or reduced potential impact on environmental disturbance due to aquacultural activities.