Microbial abundance profiling is rapidly becoming an essential method in biomedical research, though it is often costly and time intensive. We present DynaMAP, a rapid approach for microbiome profiling that uses single-molecule imaging to develop an optical map of metagenomic DNA. DynaMAP achieves strain-level taxonomic profiling without requiring base-by-base sequencing and with a one-day turnaround time. In doing so, it delivers microbiome profiling that is comparable to shotgun sequencing but operates more efficiently, strongly expanding the possibility for microbiome analysis.