Deep eutectic solvents (DES) are new ‘green’ solvents that have a high potential for biomass processing because of their low cost, low toxicity, biodegradability, and easy recycling. When Loblolly pine trees are harvested, their branches with needles are typically left in brush piles and decompose very slowly. Exploring the effect of DES pretreatment on waste pine needles was the goal of the present work. Loblolly pine needles were treated with three types of DES to prepare the biomass for enzymatic hydrolysis to glucose, a subject not readily found in the literature. The resulting products were analyzed by Fourier transform infrared spectroscopy, fiber analysis, and high-performance liquid chromatography. Glucose yields after pretreatment and hydrolysis were found to be six times that for untreated biomass with two of the DES. Fiber analysis indicated removal of lignin, hemicellulose, and ash from the needle biomass. Enhanced glucose yield was due to removal of lignin and disruption of biomass structure during pretreatment, so that the pretreated biomass was rich in cellulosic content. Based on the results shown in this study, among the three types of DES, formic acid:choline chloride and acetic acid:choline chloride pretreatment had better potential for biomass pretreatment compared to lactic acid:choline chloride.