Microbes play critical roles in the cycling of nutrients in forest ecosystems. Many studies have shown that the application of organic fertilizer can alter bacterial diversity; however, the responses of soil microbial functional genes to organic fertilization (particularly biogas slurry fertilization, or in combination with biochar) have not been elucidated as yet. We examined the responses of the abundance, diversity, and composition of soil microbial functional genes under five-years of fertilization. This fertilization involved low and high application rates of biogas slurry, at 250 and 375 m3 ha−1 yr−1, respectively, or in combination with low and high application rates of biochar, at 80 and 120 t ha−1, respectively, in a coastal poplar plantation of Eastern China. Compared to the control, the normalized signal intensity of genes in the major functional categories increased significantly in response to the quantity of applied biogas slurry, but insignificantly in response to the additional application of biochar. Subsequent to the application of biogas slurry, the functional gene evenness and Shannon index increased significantly; however, the addition of biochar to the biogas slurry had insignificant effects on the α-diversity of functional genes. These functional gene compositions under five different treatments were well distinguished, primarily through the application of biogas slurry. Our results revealed that the abundance of a large number of key functional genes involved in C, N, and P cycling were effectively increased, and these changes were well correlated with most soil properties. This study reveals the molecular level mechanisms that promote plant growth following organic fertilizer application, through the enhancement of soil microbial functional gene diversity.