Microbial community composition is essential for aquatic ecosystem functions and has been explored across diverse environments and various spatial scales. However, documented patterns are often based on samples from spatially/geographically separated locations or sites. Here, we define sampling volume as spatial scale and examine (by Illumina 16S rRNA sequencing) microbial community composition over a scale of 1 mL to 10 L in an acid mine drainage. β-Diversity analysis revealed that all samples grouped very tightly according to spatial scales and variations between every two scales were significant. Notably, mean β-diversity within each group was negatively correlated with spatial scales, indicating patchy microbial distribution. Partition of β-diversity further revealed that it was the relative abundances of some microbial taxa that largely changed among spatial scales. Phylogenetic analysis showed that microbial lineages were not randomly distributed, but displayed a tendency of more phylogenetically clustering at smaller spatial scales. Thus, we documented fine-scale spatial patterns in microbial community composition within a continuous aquatic environment, which may have practical implications for adequate sampling of aquatic systems in future studies.