While natural floods play a crucial role in shaping the composition of aquatic communities, the most rivers worldwide are regulated or dammed for anthropogenic purposes, resulting in alterations to the biological and chemical composition of these ecosystems. Studies have demonstrated various negative effects of river regulation on aquatic invertebrate communities globally. However, there is a scarcity of research in Africa, despite its vulnerability to anthropogenic impacts. This study aimed to compare aquatic invertebrate communities in the Phongolo River, an impacted regulated river, and the Usuthu River, a less impacted unregulated river, in South Africa. It further aimed to ascertain whether Lake Nyamithi, a naturally saline lake receiving water from both of the aforementioned systems, exhibited a stronger similarity to one of the two rivers in terms of its aquatic invertebrate composition. Aquatic invertebrate and water samples were collected from 2012 to 2018 over several surveys. The Usuthu River demonstrated a diverse and sensitive aquatic invertebrate community, emphasising its high conservation value. The Phongolo River demonstrated effects of anthropogenic impact, with taxa more resilient to changes in water quality and flow compared to the Usuthu River. Mismanagement and excessive water use may lead to the loss of any remaining sensitive aquatic invertebrate communities in this river. The presence of invasive molluscan in the Phongolo River and Lake Nyamithi also poses a threat to the native aquatic invertebrate communities. These invasive species are currently absent from the Usuthu River although other invasive species, such as the Australian redclaw crayfish, are found in both river systems. Lake Nyamithi displayed a unique aquatic invertebrate community, distinct from both rivers and their floodplains. This study provides important baseline information on the Usuthu River's aquatic invertebrates and emphasises the need to maintain adequate water flow in rivers and floodplains to protect biodiversity and sensitive species.