The Magellan Subantarctic ecoregion (MSE) in the Southern Hemisphere (47°-56°S; 71°-73°W) is a unique natural laboratory subject to persistent and accelerated glacial ice melt, generating a complex system of environmental gradients (e.g., salinity and temperature) that influence the ecological patterns of marine biodiversity. However, the factors influencing marine epiphytic microalgal assemblages are still poorly understood. In this context, we characterized the richness and structure of epiphytic assemblages in different benthic macroalgal hosts (Acrosiphonia arcta, Ectocarpus siliculosus, and Leptosiphonia brodiei) in sites with glaciers and estuarine characteristics (Yendegaia Bay and Fouquet Estuary) and sites without glaciers and oceanic characteristics (Batchelor River and Offing Island) of the MSE, revealing how sites, host, and environmental variables influence variation of epiphytic assemblages. In 36 samples, 67 genera of epiphytes were recorded. The dominant divisions were Bacillariophyta (50 genera), Dinophyta (7 genera) and Cyanophyta (6 genera). We observed significantly high diversity in epiphytic assemblages, with contrasting patterns of variation depending on site and/or host macroalgae. Host specificity was not evident for most epiphytes. The most factor influencing the variation of the epiphythic assemblage was the marked environmental gradient (changes in temperature, salinity, nutrients, among others) between sites with and without glacial influence. Additionally, our research identified potentially toxic and/or harmful epiphytic microalgae belonging to the divisions Dinophyta (dinoflagellates) and Cyanophyta (cyanobacteria). The data on ecological patterns of epiphyte assemblages provides valuable insights into the current state of a poorly understood microscopic biodiversity, shaped by diverse environmental factors at different sites. Under current and future climate change scenarios in the MSE, environmental gradients may become more pronounced, with important positive and/or negative consequences on epiphyte assemblages. In light of these findings, we present a baseline for future research to further develop our understanding and facilitate the monitoring and conservation of epiphytic microalgae in the MSE.