In this study, quantitative and qualitative microbiological parameters were evaluated to detect differences related to soil and crop management. The study was carried out in a field experiment installed 14 years ago on a Rhodic Eutrudox, in Londrina, PR, Brazil. Treatments were a combination of a crop sequence (S) (soybean/wheat) and a crop rotation (R) (lupin/maize/black oat/soybean/wheat/soybean/wheat/soybean), either under conventional tillage (CT) or no-tillage (NT). Evaluations were performed when all systems were under the soybean cropping season, at full flowering. Amounts of microbial biomass carbon and nitrogen (MB-C and MB-N, respectively) were 114 and 157% higher in NT than in CT. Furthermore, the metabolic quotient (qCO(2)) was lower under NT, indicating higher metabolic efficiency of the soil microbes. These parameters were not affected by the crop sequence/rotation. Genetic diversity of the total soil bacterial community was higher under NT and lower in the CT system with crop sequence. Regarding the biological N-2 fixation, it was found that plant biomass, total N and fraction of N-ureides in shoots, as well as nodule efficiency, were higher under NT. Genetic diversity of rhizobia was affected mainly by crop management and was higher under crop rotation, probably due to the greater number of plant species. However, crop rotation decreased the efficiency of the biological N-2 fixation process, which may be related to more abundant N in the soil or to a lower selection pressure for efficient rhizobia. For soil microbes with specific functions, e.g., rhizobia, genetic diversity may therefore differ from functionality.