An improved in situ hybridization approach (Polygold‐FISH) using biotinylated probes targeting multiple locations of the 16 S ribosomal subunit, followed by fluoronanogold‐streptavidin labeling and autometallographic enhancement of nanogold particles was developed as a means of signal amplification of metallo‐labeled cells, without the need for Catalyzed Reporter Deposition (CARD). Bacterial cells were readily detected based on their gold‐particle signal using scanning‐electron microscopy and energy‐dispersive X‐ray spectroscopy when contrasted with controls or cells hybridized with a single probe. Polygold‐FISH presents an alternative to CARD‐FISH, circumventing the need for aggressive oxidants, which is useful when products of microbial respiration such as those relevant at the microbe‐mineral interface could be altered during processing for visualization.