Polyacrylonitrile (PAN) nanofibers have extensive applications as filters in various fields, including air and water filtration, biofluid purification, and the removal of toxic compounds and hazardous pollutants from contaminated water. This research focuses on investigating the impacts of annealing on the mechanical and thermal characteristics of oriented PAN nanofibers produced through the electrospinning of a PAN solution. The nanofiber mats were subjected to annealing temperatures ranging from 70 ◦C to 350 ◦C and characterized using a tensile test machine, thermogravimetry, differential scanning calorimetry, and scanning electron microscopy (SEM). The study aimed to examine the tensile strength in the transverse and longitudinal directions, Young’s modulus, and glass transition temperatures of PAN nanofiber mats. The results indicate that, upon annealing, the diameter of the nanofibers decreased by approximately 20%, while the tensile strength increased in the longitudinal and transverse directions by 32% and 23%, respectively. Furthermore, the annealing temperature influenced the glass transition temperature of the nanofiber mats, which exhibited a 6% decrease at 280 ◦C, while the degradation temperature showed a slight increase of 3.5% at 280 ◦C. The findings contribute to a better understanding of the effects of annealing on PAN nanofiber mats, facilitating their potential for various filtration applications.