Film mulching and N fertilization can affect soil physicochemical properties, thereby improving plant growth, and may in turn affect soil microbial communities. Therefore, a 2-year field experiment was conducted to research the effects of film mulching and N fertilization on soil microbial communities. The four main treatments were N0F0, N0F1, N1F0, and N1F1, combining two N fertilizer rates (N0, 0 kg N ha−1; N1, 225 kg N ha−1) and two mulching methods (F0, no mulching; F1, film mulching) in the absence and presence of plants. The film mulching treatments significantly increased the mean temperature by 0.2 °C and decreased the soil organic carbon (SOC), mineral N and water soluble organic C by 5.6%, 35.5% and 24.0%, respectively. The N fertilization treatments significantly increased the mineral N, water soluble organic N and KMnO4-oxidizable C by 117.9%, 256.4% and 55.3%, respectively. Additionally, the phospholipid fatty acid (PLFA) analysis of the soil microbial community revealed that the film mulching treatments significantly decreased the total PLFAs by 21.5% and the absolute abundance of fungi (F), bacteria (B), and actinomycetes by 26.7%, 23.1% and 24.6%, respectively. N fertilization significantly decreased the Gram-positive B/Gram-negative B ratio by 9.8%. Film mulching combining N fertilization significantly decreased the F/B ratio by 10.0%. Temperature (P < 0.001) and SOC/total P (P < 0.001) were confirmed to play significant roles in shaping the soil microbial community. Accordingly, short-term film mulching increases soil organic matter decomposition in the top soil and decreases the total soil microbial biomass and most microbial communities.