The effects of crop manure and inorganic fertilizers on composition of microbial communities of central high land soils of Kenya are poorly known. For this reason, we have carried out a thirty-two-year-old long-term trial in Kabete, Kenya. These soils were treated with organic (maize stover (MS) at 10 t ha(-1), farmyard manure (FYM) at 10 t ha(-1)) and inorganic fertilizers 120 kg N, 52.8 kg P (N(2)P(2)), N(2)P(2) + MS, N(2)P(2) + FYM, a control, and a fallow for over 30 years. We examined 16S rRNA gene and 28S rRNA gene fingerprints of bacterial and fungal diversity by PCR amplification and denaturing gradient gel electrophoresis separation, respectively. The PCR bacterial community structure and diversity were negatively affected by N(2)P(2) and were more closely related to the bacterial structure in the soils without any addition (control) than that of soils with a combination of inorganic and organic or inorganic fertilizers alone. The effect on fungal diversity by N(2)P(2) was different than the effect on bacterial diversity since the fungal diversity was similar to that of the N(2)P(2) + FYM and N(2)P(2) + MS-treated. However, soils treated with organic inputs clustered away from soils amended with inorganic inputs. Organic inputs had a positive effect on both bacterial and fungal diversity with or without chemical fertilizers. Results from this study suggested that total diversity of bacterial and fungal communities was closely related to agro-ecosystem management practices and may partially explain the yield differences observed between the different treatments.