The experiment was aimed to investigate the effects of ENR (enrofloxacin) on microbial community diversity in soil. The molecular diversity and community structure of nitrogen-fixing microbes were analyzed by PCR-based cloning and RFLP (restriction fragment length polymorphism) approach. PCR-RFLP analysis revealed that the percentages of OTUs to total clones among groups Ⅰ to Ⅵ were 34.31%, 32.18%, 26.04%, 20.83%, 19.09% and 20.00%, respectively after 35 days′ incubation, and that the percentages of them were 23.85%, 20.75%, 18.26%, 16.67%, 14.58% and 11.67% after 70 days′ incubation, respectively. The effects of ENR on molecular diversity of nitrogen-fixing microbial communities were enhanced with the increasing ENR concentrations. The results showed that the Margalef Index in CK was significantly (P<0.05) higher than that in the soils in which ENR was added after 35 days. However, the Margalef Index in CK was only significantly higher than that in the soils treated with 10 g/g and 50 g/g of ENR after 70 days, respectively. As for the Shannon-Wiener Indexes, CK was significantly higher than those in the groups treated with ENR except for 0.01 g/g after 35 days. The Shannon-Wiener Indexes were the same as Margalef Indexes after 70 days. It was indicated that the effects of ENR on molecular diversity of nitrogen-fixing microbial communities in the treated groups with ENR at the concentrations of 0.01 g/g to 1 g/g were not significantly distinguished from that of CK with the increasing of treatment time.