Woody plant encroachment is a globally-prevalent vegetation change phenomenon that has shifted grass-dominated ecosystems to mixed grass and woody plant matrices over the last century. In the Rio Grande Plains of Texas, the introduction of N-fixing woody legumes has increased above- and belowground primary productivity and changed the litter chemistry of the system, accelerating rates of belowground biogeochemical processes. The purpose of this study was to assess the impact of grassland to woodland transition on i) P concentrations in soil physical fractions that differ in their organic matter turnover rates, ii) P availability within the soil over the course of woody encroachment and across the landscape, and iii) microbial community composition and diversity. Soil samples were collected in remnant grasslands and four woody landscape elements (clusters, groves, drainage woodlands, and playas) along a 135-yr chronosequence of woody plant encroachment. P was fractionated by the Hedley method and P concentrations were determined by alkaline oxidation and lithium fusion coupled with ascorbic acid colorimetry. Bacterial and fungal communities were characterized by molecular methods. Whole soil P concentrations were 2-5X greater in woody landscape elements than in grasslands, and nutrient concentrations increased linearly with time following woody plant invasion in all but the slowest-cycling physical fractions. Plant-available P and organic P increased dramatically with time following encroachment. Changes in P availability were more pronounced in drainages and playas than in upland clusters and groves. Analysis of the bacterial and fungal communities demonstrated that microbial communities in grasslands differ at both phylum and genus level from the flora of the wooded landscape elements. This study demonstrates that woody encroachment strongly influences the distribution and availability of soil P and indicates that nutrient cycles in the soil are closely linked and similarly affected by increased woody plant abundance. Microbial communities under woody species differ in composition from those of the grasslands, and are likely contributing to the observed changes in nutrient availability. Since N and P are generally the most limiting nutrients in terrestrial ecosystems, increased stores of P are likely to alter rates of microbial processes, plant-microbe and plant-plant interactions, and successional dynamics in this ecosystem and similar landscapes around the world.