Ecological integrity, which encompasses both functional and structural integrity, provides a framework for assessing the health of ecosystems impacted by environmental stressors linked to human activities. Leaf-litter breakdown is a key indicator of the functional integrity of stream ecosystems and cotton strips have been employed as a standard substrate for quantification of decomposition rates in streams across a broad range of spatial scales. Cotton strips can also be used to assess the composition of stream microbial communities involved in decomposition as an indicator of stream structural integrity. A key assumption of this approach is that microbiomes that colonize cotton are similar to those that colonize leaves, but this assumption has not been explicitly tested. We confirmed via field incubations in an urban river that cotton strips were colonized by bacterial and fungal assemblages that did not differ in species richness and diversity from tree leaves. Cotton microbiomes included bacterial and fungal orders that also colonized leaves and are associated with leaf detritus in streams, and the similarity between cotton and leaf microbiomes increased during incubation in the stream. However, cotton was also colonized by a greater abundance of cellulose-degrading bacteria and fungi than leaves. These results support use of cotton strips to assess stream structural integrity, and we conducted a laboratory-scale stream mesocosm experiment to demonstrate the application of cotton strips to assess the effect of pharmaceuticals on stream functional and structural integrity.