The effects of modifying electrospun poly(vinyl alcohol) (PVA) nanofibers through crosslinking using glutaraldehyde (GA) are explored in this paper. Various concentrations of PVA solutions containing model drugs rifampicin (RIF) and isoniazid (INH) were electrospun and thereafter crosslinked using GA vapors. PVA nanofibers demonstrated high drug entrapment efficiency of 98.77% ± 1.384% and 95.07% ± 1.988% for the INH- and RIF-loaded PVA nanofibers, respectively. The surface morphology, molecular vibrational transitions, tensile attributes and in vitro drug release were characterized and supported by in silico molecular mechanics simulations. Results indicated that crosslinking caused a significant reduction in the rate of drug release where 81.11% ± 2.35% of INH and 59.31% ± 2.57% of RIF were released after 12 h. Tensile properties such as the ultimate strength and Young's modulus increased after crosslinking, caused by crosslinks forming between PVA nanofibers as was revealed through scanning electron microscopy analysis. Fourier Transform infrared analysis was conducted to further support the mode of crosslinking. Additionally, image processing analysis was carried out to quantify the effect of formulation variables on the morphology of nanofibers. Furthermore, the effect of GA-induced crosslinking and addition of drugs on the performance of electrospun fibers was further elucidated and conceptualized using a molecular mechanics assisted model building and energy refinement approach via molecular mechanics energy relationships by exploring the spatial disposition of energy-minimized molecular structures of the polymer, crosslinker and the drugs.