Smart auxetic structures are gaining attention in various areas such as architecture, clothing (sports and protective), civil, and medical applications owing to their negative Poisson’s ratio. Compared to ordinary structures, these structures have better properties (shear resistance, formability, energy absorbance, and robust fracture strength). Auxetic structures show the exceptional property of becoming wider in one direction when stretched from another direction. In this research, three different auxetic weft-knitted structures were fabricated using nylon, polyester, acrylic, and cotton yarns on a Shima Seiki flat-knitting machine. The physical properties, negative Poisson’s ratio, and thermo-physiological comfort properties of these fabrics were checked. Negative Poisson’s ratio strain curves of the developed fabrics were plotted; all fabrics, except for nylon, show the negative Poisson’s ratio (NPR). The NPR decreases with increased strain in the longitudinal direction, and polyester exhibits a maximum value of NPR −0.4 in line structure at 30 mm extension. Results also revealed that structures made with nylon and polyester yarns exhibit a better value of air permeability than acrylic and cotton, while acrylic provides the best thermal resistance values than other materials in line structure and polyester yarn shows better overall moisture management capacity (OMMC) performance in zigzag structures.