In a pot-cultural experiment, the impact of the antibiotic roxithromycin (ROX) addition was assessed on the diversities of microbial structure and function communities, especially involved in ammonia and nitrite oxidation in wheat rhizosphere soil with and without the addition of earthworms. The abundances of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and total bacteria were surveyed by the quantitative PCR. The quantities of total bacteria, AOB, and NOB with earthworms were higher than those without earthworms because of the synergistic effect. ROX inhibited the growth of AOB in all treatments, although the quantities of AOB were in a light increase in medium and heavy polluted treatments compared with that in the light polluted treatments. Different from AOB, the quantities of NOB were lowest in light polluted treatments, but the quantities of NOB were rapidly increased in medium and heavy polluted treatments compared with that in the control. These results indicated that the application of ROX principally had a negative effect on nitrification performance by affecting the abundances and relative ratios of both AOB and NOB in soil communities, which affected the N cycle in an agricultural ecosystem. According to the metabolic diversities evaluated by the biologic assay, the tendency of metabolic diversities was quite contrary to the quantities of NOB in all treatments and showed the contrast growing relation of autotrophic and heterotrophic bacteria under ROX pollution pressure in agricultural ecosystems.