The effects of 3 and 19 yr natural fallows on soil structure and soil bacteria were assessed by aggregate size fractionation of a tropical sandy soil (0–10 cm). Bulk unfractionated soil and fractions including particulate coarse organic residues (COR), and four fractions (>2000, 50–2000, 2–50 and 50 μm), suggesting that these microhabitats were favourable to active N2 fixers. In contrast, more than 70% of the N2 fixing microorganisms and 90% of the recovered Azospirillum were isolated from the dispersible clay fraction (0–2 μm). Diversity of Azospirillum species was assessed by hybridisation with specific genetic probes on colonies within each fraction. This approach revealed the abundance of Azospirillum irakense in the 3 yr fallow soil fractions only and a selective effect of fallow on Azospirillum brasilense/Azospirillum amazonense genomic species in the 19 yr fallow soil. These changes would remain hidden, if the investigation had been restricted to the unfractionated soil.