Electrospinning has emerged as a potential method to fabricate nonwoven nanofibers. It has application in different fields of biomedicine as it has potential to carry antimicrobial and bioactive agents. The present investigation was conducted to optimize the process conditions and determine the viability of probiotics after being electrospun in fibers. Poly(vinyl alcohol) (PVA) was utilized as electrospun material because it possesses generally recognized as safe (GRAS) status and in dry form it acts as a high oxygen barrier and has high water solubility. This characteristic allows the easy recovery of the bacteria from electrospun fibers. The viability tests, carried out at three different temperatures (room temperature, 4°C and -20°C) showed Bifidobacterium animalis subsp. Lactis Bb12 (probiotic 1) and combination of Streptococcus thermophilus (TH-4®), Lactobacillus paracasei 431® and Bb-12 (probiotic 2) within the electrospun PVOH fibers remained viable after 1 week at room temperature and refrigeration temperature. The nanofibers containing probiotics prepared with 9% poly venyl alcohol showed homogenous, uniform, bead-free and smooth texture. Probiotic 1 demonstrated growth as 1.85×108, 1.57×108 and 1.71×108 before, 0 hour and after 1 week of encapsulation. While probiotic 2 exhibited a growth of 2.1×108 before electrospinning, 1.3 ×108 at 0 hour and 1.97×108 after one week of electrospinning. There was no change in CFU/mL count and remained 108 CFU/mL. The encapsulation efficiency was 84.07% and 85.73% at 0 and one week, respectively, for Probiotic 1, while probitic 2 showed 90.09% and 93.59 % encapsulation efficiency before and after one week, respectively. Considering the prolonged viability of nanofibers containing probiotics noted at room temperature, this technology can be implemented for prolonged viability of probiotics.