The overall impact of tuna farming on soft-bottom habitat was assessed at three tuna farms over a period of 3 years, using benthic macroinvertebrates as indicators. Polychaetes and amphipods served as better indicators of the impact of tuna farming compared with molluscs and decapods. Lower number and Shannon–Wiener diversity of polychaete and amphipod taxa were recorded over time at the impacted plots compared with the control plots, while the polychaete/amphipod index indicated that the Ecological Quality Status at the impacted plots changed from “Poor”/”Moderate” to “Good” during the study period. Results of the multivariate analyses indicated significantly higher dispersion of samples of the polychaete and amphipod assemblages over time at the impacted plots compared with the control plots, indicative of stressed assemblages. Differences in the macroinvertebrate assemblages between impacted and control plots were consistent across faunal groups except for molluscs, which showed no response. Results must be interpreted with caution due to the high spatiotemporal variation in the influence of tuna farming on the macroinvertebrate assemblages, which highlights the importance of including multiple impacted and reference areas, as well as replicate sampling times, in assessing the environmental impact of tuna farms.