The quantity and/or quality of soil organic matter (SOM) and its fractions regulate microbial community composition and associated function. In this study an established, replicated agricultural systems trial in a semi-arid environment was used to test: (i) whether agricultural systems which have increased plant residue inputs increase the amount of labile SOM relative to total SOM, or change the quality of SOM fractions; and (ii) whether the size or quality of OM fractions is most strongly linked to the size, activity, functional diversity, and community structure of the soil microbial biomass. Soil (0-50 mm) was collected following 5 years of continuous wheat, crop rotation, crop-pasture rotation, annual pasture, or perennial pasture. Pastures were grazed by sheep. Direct drilling and non-inversion tillage techniques were compared in some cropping systems. Total carbon (C) increased with the proportion of pasture as a result of increased SOM inputs into these systems; land use also significantly affected SOM fractions and their chemical and physical nature. While the size, function, and structure of the soil microbial community were somewhat related to total soil C, they were better correlated with SOM fractions. The C : nitrogen (N) ratio of light fraction organic matter could be used to predict the amount of potentially mineralisable N in soil, while the C : N ratio of total SOM could not. Measurement of bacterial community structure (using denaturing gradient gel electrophoresis) significantly discriminated between land uses, while community-level physiological profiles revealed fewer differences. Overall, our findings support the premise that labile fractions of SOM are more strongly related to microbial community structure and function than is total SOM.