Abstract: Soil microbial biomass carbon and nitrogen (SMBC and SMBN, respectively) are key factors that characterize soil fertility and its’ change. The rhizosphere is a hotspot of microbial interactions in rice fields. Rhizosphere microbiota are important for rhizosphere ecology and are the impetus of soil organic matter and nutrient transformation. This study investigated bulk and rhizosphere SMBC and SMBN in paddy soils in response to wheat straw addition and different amounts of nitrogen fertilizer application in the typical rice and wheat rotation areas of the middle and lower reaches of the Yangtze River. There were four treatments (in triplicate): no straw or nitrogen fertilizer (CK), straw addition (SN0), straw and low nitrogen fertilizer addition (SN1), and straw and high nitrogen fertilizer addition (SN2). The results showed that, compared to CK, the SMBC contents in the SN0 rhizosphere and bulk of high sandy soil increased by 40.3% and 48.1%, respectively, while those in yellow mud soil increased by 95.7% and 75.4%, respectively. The SMBN contents in the rhizosphere of high sandy soil did not change significantly, but decreased by 19.9% in bulk soil. The SMBN contents in the rhizosphere and bulk of SN0-treated yellow mud soil decreased by 19.5% and 49.0%, respectively. Low nitrogen fertilizer application significantly increased the SMBC content in the rhizosphere of sandy soil and the bulk of yellow mud soil and increased the SMBN content in the rhizosphere and bulk of both soils. With increased nitrogen fertilizer application, SMBC and SMBN contents in the rhizosphere and bulk of both soils significantly increased. Compared with SN0, the SMBC content in the rhizosphere of SN1 in sandy soil increased by 5.1%, that in bulk decreased by 12.9%, and that in the bulk of yellow mud soil increased by 11.1%. There was no significant change in SMBC content in the rhizosphere of yellow mud soil. SN1 treatment led to an increase in the SMBN contents of the rhizosphere and bulk of sandy soil by 17.3% and 9.8%, respectively, and an increase in the SMBN contents of the rhizosphere and bulk of yellow mud by 36.1% and 68.9%, respectively. SN2 treatment led to an increase in the SMBC content in the sandy soil rhizosphere and bulk by 8.6% and 39.3%, respectively, and by 34.58% and 3.05% in yellow mud, respectively, over those of SN0. For the SN2 treatment, sandy soil rhizosphere and bulk SMBN increased by 27.0% and 13.5%, respectively, and they increased by 25.6% and 232.9%, respectively, in yellow mud soil, compared with SN0. These comprehensive analyses show that nitrogen fertilizer addition can increase the SMBC and SMBN contents in bulk and rhizosphere soils, thereby improving soil nutrient availability. Straw returning with nitrogen fertilizer greatly improves soil fertility and promotes crop growth in the rice-wheat rotation areas of the middle and lower reaches of the Yangtze River.