ABSTRACT Microscopic tubules and filaments composed of iron minerals occur in various rock types of all ages. Although typically lacking carbonaceous matter, many are reasonably interpreted as the remains of filamentous microorganisms coated with crystalline iron oxyhydroxides. Iron‐oxidizing bacteria (IOB) acquire such a coating naturally during life. However, recent debates about purported microfossils have highlighted the potential for self‐organized nonbiological mineral growth (particularly in chemical gardens) to form compositionally and morphologically similar tubules. How can biogenic and abiogenic iron‐mineral tubules be differentiated? Here, we use optical and electron microscopy and Mössbauer spectroscopy to compare the composition, microtexture, and morphology of ferruginous chemical gardens and iron‐mineralized sheaths of bacteria in the genus Leptothrix . Despite broad morphological similarity, we find that Leptothrix exhibits a narrower range of filament diameters and lower filament tortuosity than chemical gardens. Chemical gardens produced from a ferrous salt also tend to incorporate Fe 2+ whereas Leptothrix sheaths predominantly do not. Finally, the oxyhydroxides formed in Leptothrix sheaths tend to be smoother and denser on the inward‐facing side, rougher and sparser on the outward side, whereas for chemical garden tubules the reverse is true. Some of these differences show promise for the diagnosis of natural samples.