Palace Leas, a long-term experiment at Cockle Park Farm, Northumberland, UK was established in winter 1896–1897 since when the 13 plots have received regular and virtually unchanged mineral fertiliser and farm yard manure inputs. Fertilisers have had a profound impact on soil pH with the organically fertilised plots showing a significantly higher pH than those receiving mineral fertiliser where ammonium sulphate has led to soil acidification. Here, we investigate the impact of organic and mineral fertilisers on the actinobacterial community structure of these soils using terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene analysis. To differentiate fertiliser effects from seasonal variation, soils were sampled three times over one growing season between May and September 2004 and January 2005. Community profiles obtained using T-RFLP were analysed using multivariate statistics to investigate the relationship between community structure, seasonality and fertiliser management. Soil pH was shown to be the most significant edaphic factor influencing actinobacterial communities. Canonical correspondence analysis, used to investigate the relationship between the 16S rRNA gene community profiles and the environmental parameters, showed that actinobacterial communities also responded to soil water content with major changes evident over the summer months between May and September. Quantitative PCR of the actinobacterial and fungal 16S and 18S rRNA genes, respectively suggested that fungal rRNA gene copy numbers were negatively correlated (P = 0.0131) with increasing actinobacterial signals. A similar relationship (P = 0.000365) was also evident when fatty acid methyl esters indicative of actinobacterial biomass (10-methyloctadecanoic acid) were compared with the amounts of fungal octadecadienoic acid (18:2ω9,12). These results show clearly that soil pH is a major driver of change in actinobacterial communities and that genera such as Arthrobacter and Micrococcus are particularly abundant in soils receiving organic inputs whilst others such as Streptomyces, Acidimicrobium and Actinospica are more prevalent in acid soils. The importance of these findings in terms of fungal abundance and potential disease suppression are discussed.