Agricultural management can affect soil organic matter chemistry and microbial community structure, but the relationship between the two is not well understood. We investigated the effect of crop rotation, tillage and stubble management on forms of soil phosphorus (P) as determined by solution 31P nuclear magnetic resonance spectroscopy and microbial community composition using fatty acid methyl ester analysis in a long-term field experiment (26 years) on a Chromic Luvisol in New South Wales, Australia. An increase in soil organic carbon, nitrogen and phosphorus compared to the beginning of the experiment was found in a rotation of wheat and subterranean clover with direct drill and mulching, while stubble burning in wheat-lupin and wheat-wheat rotations led to soil organic matter losses. Microbial biomass was highest in the treatment with maximum organic matter contents. The same soil P forms were detected in all samples, but in different amounts. Changes in organic P occurred mainly in the monoester region, with an increase or decrease in peaks that were present also in the sample taken before the beginning of the experiment in 1979. The microbial community composition differed between the five treatments and was affected primarily by crop rotations and to a lesser degree by tillage. A linkage between soil P forms and signature fatty acids was tentatively established, but needs to be verified in further studies.