Polylactic acid (PLA) fiber is a degradable material with good environmental friendliness for textile applications. However, the main problems of difficult dyeing of PLA fibers were: high crystallinity to the adsorption of dyes, more ester and methyl groups producing non-hydrophilic problems, long chains making dyes difficult to penetrate, and producing a low dyeing rate. Here, we attempted to change the crystallinity of the PLA fiber to a lower degree from hydrophobic to hydrophilicity property variation, destroy the long chain structure to grant more staining sites, and improve the PLA fiber staining depth and the resilience dyeing effect with deep eutectic solvent (DES) treatment and natural dyes. We discovered that a controlled DES treatment process could make PLA fibers less crystallized, help amorphous areas form, and break up long chains, which lead to more dye sites. After DES treatment, the crystallinity decreased from 56.12 to 29.86%, and the instantaneous water contact angle decreased from 108.79 to 64.39°. The DES-treated PLA fabric exhibited a higher K/S value of 15.14 for natural dyes under specific conditions. The fabric, which had remarkable fastness characteristics and wash resistance, could endure frequent laundering and fulfill the demands of everyday use. Moreover, the fabric had good antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans and possessed a certain level of biocompatibility with fibroblasts. This DES treatment and natural dye combination method offered a new strategy for improving PLA fabric staining depth and color fastness, making it a promising option for low-carbon environmental protection in the textile industry.