Macroplastic pollution remains a growing global environmental concern, and our understanding of its interaction with aquatic organisms is underdeveloped. It is also less clear how hydraulic biotopes influence macroinvertebrates colonisation of macroplastic relative to natural substrates. We investigated temporal and spatial patterns of macroinvertebrate colonisation on macroplastic litters in contrasting stream hydraulic biotopes (riffle, pool, and run) in minimally impacted headwater streams of Eastern Cape, South Africa. Plastic substrates of different proportions of natural and plastic litter were deployed across four sites. The Substrate group included 100% natural substrates (NS), 50% natural material and 50% plastic litters (NP), and 100% plastic (PD) litters. Each substrate group was deployed in riffle, pool, and run habitats for six months at each site. Across hydraulic biotopes, macroinvertebrate colonised substrate groups equally (PERMANOVA, p > 0.05). Macroinvertebrate diversity indices were statistically different across substrate groups in pools but not in other hydraulic habitats (PERMANOVA, p < 0.05). We observed that NS had significantly higher macroinvertebrate Margalef’s richness, Shannon, and Simpson diversity values than macroplastic substrates in pools. This difference suggests that specific-hydraulic biotope characteristics, such as sediment accretion and stream discharge, influence macroinvertebrate diversities. However, the dominant taxa had a marked presence in all substrate groups within hydraulic biotopes throughout the study, resulting in temporal variance that was not significant. Our findings highlight the importance of hydraulic biotope influence on macroinvertebrate colonisation of macroplastic substrates. It also provides a baseline for further research involving riverine macroplastic pollution.