A greenhouse pot experiment was conducted to investigate the influence of soil moisture content and plant species on soil microbial community structure using cultivation-independent methods. White clover and ryegrass were grown individually or in a mixture. Plants were subjected to soil moisture content corresponding to 60% field capacity (FC) and 80% FC. Total plant biomass of white clover and ryegrass increased with increasing soil moisture contents. At a given soil moisture content, total biomass of white clover was lower in the ryegrass–clover (RC) mixture compared with those grown individually, while total biomass of ryegrass was higher. Microbial community structure assessed by phospholipid fatty acid analysis (PLFA) was more affected by plant species than soil moisture. Community level physiological profiles (CLPP), in terms of diversity of substrate utilization and average well colour development (AWCD) were affected by plant species and soil moisture. Soil moisture effects were more pronounced in clover than in ryegrass. AWCD and diversity of substrate utilization in the ryegrass–clover mix were similar to those of sole clover while they differed from that of ryegrass suggesting a dominant effect of clover in the mix.