In stream ecosystems, periphyton is a hotspot of microbial diversity and biochemical cycling driven by essential metabolic and enzymatic activities. Hydrologic intermittence leads to highly dynamic landscapes, shaping periphyton microbial composition and interplay. Here, we describe the microbial communities (bacteria, phototrophs, and fungi) in the periphyton of the Cube River network along a spatiotemporal gradient of hydrologic intermittence by a metagenomic approach in six sampling campaigns during 2021. Our findings suggest that local conditions highly influence microbial diversity. Hence, the abundance of bacteria, phototrophs, and fungi depends on seasonality and the proportion of intermittent reaches across the river network, mostly headwaters. Most of the differentiation in the diversity of these groups is driven mainly by changes in the availability of major dissolved water constituents (e.g., C, N, P). Bacteria dominate the consortium, but its abundance decreases with dry conditions, while algae tend to increase with dry phases, and fungi remain relatively similar and abundant throughout the hydrological regime. We also found that phototrophs might be crucial in shaping bacterial and fungal composition. Interactions among these three main groups increase in complexity as hydrologic intermittence intensifies. Overall, our findings shed unknown microbial dynamics on stream periphyton in an intermittent river network in the Ecuadorian Chocó, a global hotspot of diversity with important ecosystem functions and highly vulnerable to climate change and anthropogenic activities.