Simple Summary Developing a sustainable agroforestry system requires the utilization of byproducts. Poultry production and forestry are the two largest agricultural industries in Mississippi (USA) with loblolly pine being one of the most predominant timber species in the southeast. Poultry litter is a high-nutrient waste generated in poultry systems that is used as a fertilizer. However, poultry litter can release pollutants that reduce air and water quality and carries a heavy microbial load. Biochar and wood vinegar effects on nutrient retention and microbial activity vary due to the source material, production conditions, and application rate. This research evaluated how loblolly pine biochar and wood vinegar influence nutrients and microbial activity over time. The study found that wood vinegar reduced microbial growth in poultry litter-biochar blends. Fungi thrived in higher biochar proportions and bacteria preferred less biochar. Nitrogen and phosphorous were retained in 10% and 20% biochar blends. Furthermore, 10% and 20% biochar treatments were observed to be less odorous and compact, and the high pH of the biochar would be an effective conditioner for acidic soils. This study provides valuable information to stakeholders on how to utilize these materials to meet their objectives using regionally generated waste streams in the southeastern USA. Biochar, wood vinegar, and poultry litter are waste streams that can be utilized as soil amendments and fertilizers. However, poultry litter releases several pollutants through nutrient leaching and carries heavy microbial loads, including potential human pathogens. Improving nutrient retention and reducing microbial load in poultry litter may help protect environmental and human health and improve its value as a soil amendment. The objectives of this study were to determine how blending varying proportions of loblolly pine (Pinus taeda L.) biochar, wood vinegar, and poultry litter affected nutrient profiles and microbial abundance over time. Biochar inclusion rates were 0%, 5%, 10%, and 20%, and wood vinegar was applied at 2% w/w. Samples were taken at Day 0, 57, and 112 to measure nitrogen, phosphorus, potassium, pH, total fungi, and total bacteria. Nutrient levels generally decreased with increasing biochar level; however, biochar inclusion rates of 10% and 20% retained nitrogen and phosphorus and exhibited improved physical properties. Overall, adding wood vinegar decreased nutrient concentrations and showed a biocidal effect for bacteria and fungi. Bacteria and fungi showed different relationships with biochar inclusion rates, with fungi preferring higher biochar inclusion rates and bacteria flourishing at lower biochar inclusion rates.