Soil fertility decline is increasingly leading to reduced food production worldwide. Over 70% of small holder farmers in the central highlands of Kenya are using crop manure, animal wastes and inorganic fertilizers to increase their farms¿ fertility and subsequent productivity. The dilemma with these practices is that less is known on the impact of these resources on the below ground biodiversity particularly the microbial communities which play a key role in determining soil quality. A study was carried out on a 32 year old long-term trial in Kabete, Kenya. These soils were treated with organic (maize stover at 10 t ha-, farmyard manure at 10 t ha-) and inorganic fertilizers (120 kg N, 52.8 kg P plus farmyard manure at 10 t ha-1 (N2P2 + FYM), 120 kg N, 52.8 kg P plus maize stover at 10 t ha-1 (N2P2 + R), 120 kg N, 52.8 kg P (N2P2), and a control (Nil and fallow) for over 30 years. We examined 16S rRNA gene and 28S rRNA gene fingerprints of bacterial and fungal communities, respectively, by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. Bacterial community structure and diversity were negatively affected by N2P2, as evidenced by changes in the PCR-DGGE banding patterns. Bacterial community structure in the N2P2-treated soil was more closely related to the bacterial structure in the untreated soil (fallow and Nil) than that in soils treated with a combination of inorganic and organic or inorganic fertilizers alone. For the fungal community the negative effect of N2P2 alone was not as adverse as for the bacterial community structure since the soils treated with N2P2 were closely related to those treated with N2P2 + FYM and N2P2 + maize stover. However, soils treated with organic inputs clustered away from soils amended with inorganic inputs. Organic inputs had a positive effect on both fungal and bacterial community structures with or without chemical fertilizers. Results from this study suggested that bacterial and fungal communi