There is increasing interest in good agriculture practices that address the issues of sustainability, reduction in inputs such as fertilizers and pesticides while maintaining crop yield and soil fertility. It is important that soil microbial diversity and function are not impaired by altered agricultural practice. In this study, as indicators of soil quality, the bacterial community structure was evaluated from a long-term field trial managed with conventional and low-input fertilization/pesticide regimes. The low-input plots under study received approximately one fifth less N fertilizer than the conventional-input plots, a maximum of half the recommended application rates of fungicides and pesticides and no externally added P source. A non-culturable approach was taken using polymerase chain reaction–denaturing gradient gel electrophoresis analysis of 16S rRNA and alkaline phosphomonoesterase [phosphatase] (ALP) genes in an attempt to relate bacterial community structure to respective field management regimes. To identify the ALP bacteria in these plots, randomly selected ALP clones were sequenced. The results based on Shannon diversity indices and community structure analysis of ALP genes suggest differences in community diversity and structure under conventional and low-input barley sites in most sampling seasons. We conclude that soil fertilization management affects the ALP bacteria in the barley rhizosphere, while the overall changes in bacterial community in these sites are prominently due to seasonal variation compared to crop or input regimes. The randomly selected ALP sequences identified from these sites were mostly from the Alpha and Gammaclasses of Proteobacteria.