This paper reports a study on the comfort properties of poly(lactic acid) (PLA)/cotton multilayered quilted fabrics. Fifteen multilayered quilted fabrics with diverse structural parameters were first fabricated using three different linear densities of PLA filaments as inlaid yarns and five distinct blending ratios of PLA/cotton blended yarns as surface yarns. Then, their comfort properties, including air permeability, water vapor permeability, and thermal and compressive properties, were tested. The effects of the inlaid yarns' linear density and surface yarns' blending ratio on these properties were systematically analyzed, and multiple regression models were established to accurately describe these relationships, with all correlation coefficients exceeding 0.97. Based on this, a multi-objective optimization model was developed, and optimization calculations were performed using the MATLAB genetic algorithm tool to determine the optimal yarn parameters for enhancing overall comfort performance. The results indicated that selecting 450D PLA for the inlaid yarn and a 50/50 PLA/cotton blend for the surface yarn achieved superior overall comfort properties, along with enhanced air and moisture permeability. To improve thermal and compressive properties, it was recommended to use inlaid 900D PLA and surface 100% PLA yarns. Furthermore, these optimized fabrics exhibited excellent dimensional stability and tensile properties even after undergoing 20 washes, proving their durability. This study will promote PLA application in textiles and provide valuable insights for designing eco-friendly quilted fabrics.