Piezoelectric nanogenerators, harvesting energy from mechanical stimuli in our living environments, hold great promise to power sustainable self-sufficient micro/nanosystems and mobile/portable electronics. BaTiO3 as a lead-free material with high piezoelectric coefficient and dielectric constant has been widely examined to realize nanogenerators, capacitors, sensors, etc. In this study, polydimethylsiloxane (PDMS)-based flexible composites including BaTiO3 nanofibers with different alignment modes were manufactured and their piezoelectric performance was examined. For the study, BaTiO3 nanofibers were prepared by an electrospinning technique utilizing a sol gel precursor and following calcination process, and they were then aligned vertically or horizontally or randomly in PDMS matrix-based nanogenerators. The morphological structures of BaTiO3 nanofibers and their nanogenerators were analyzed by using SEM images. The crystal structures of the nanogenerators before and after poling were characterized by X-ray diffraction. The dielectric and piezoelectric properties of the nanogenerators were investigated as a function of the nanofiber alignment mode. The nanogenerator with BaTiO3 nanofibers aligned vertically in the PDMS matrix sheet achieved high piezoelectric performance of an output power of 0.1841 mu W with maximum voltage of 2.67 V and current of 261.40 nA under a low mechanical stress of 0.002 MPa, in addition to a high dielectric constant of 40.23 at 100 Hz. The harvested energy could thus power a commercial LED directly or be stored into capacitors after rectification.