AbstractMicroorganisms can have a substantial effect on labile phosphorus (P), which may be lost from the soil surface and impact water quality. Changes in nutrient availability and soil health from agricultural management can affect microbial biomass carbon (MB‐C), microbial biomass phosphorus (MB‐P), and the expression of P cycling enzymes. The objective of this research was to investigate biological mechanisms affecting P availability and potential loss to runoff in a no‐till, corn (Zea mays)–soybean (Glycine max) cropping system with two cover crops (cover crop [CC] and no cover crop [NC]) and three P management treatments (fall broadcast [FB], spring injected ammonium polyphosphate [SI], and no phosphorus application [NP]). Treatments were applied to a randomized block design with three replicates. Soil samples were analyzed for MB‐P, MB‐C, phosphatase activity, and Mehlich‐III P (PM). The P supply to the soil solution was measured using diffusive gradient thin film P (PDGT). In Spring 2018, Fall 2018, and Spring 2019, all phosphatase activity was greater in CC versus NC (p < 0.01). Microbial biomass C was greater in CC compared with NC in spring but not fall samplings. On average, MB‐P was fivefold greater in the P fertilized than unfertilized treatments (p < 0.001). CCs did not change MB‐P, PM, or PDGT within FB or NP, but did affect SI fertilizer treatments. Our results suggest CC can increase potential for organic P mineralization, application of P fertilizer increases MB‐P, and an interaction between SI P fertilizer management and CC may increase P supply to the soil solution.