In this study, optimized cephalexin-loaded niosomal formulations based on span 60 and tween 60 were prepared as a promising drug carrier system. The niosomal formulations were characterized using a series of techniques such as scanning electron microscopy, Fourier transformed infrared spectroscopy, dynamic light scattering, and zeta potential measurement. The size and drug encapsulation efficiency are determined by the type and composition of surfactant. The developed niosomal formulations showed great storage stability up to 30 days with low change in size and drug entrapment during the storage, making them potential candidates for real applications. Moreover, the prepared niosomes showed negligible cytotoxicity for HepG2 cells, measured by MTT assay. The antibacterial properties of cephalexin-loaded niosome were investigated using S. aureus and E. coli as gram-positive and gram-negative bacteria, respectively. The results showed that the encapsulation of antibiotic drug in niosomal formulation could enhance the antibacterial efficiency of the drug, where the minimum inhibitory concentration was droped from 8 µg/mL (cephalexin) to 4 µg/mL (cephalexin-loaded niosome) and from 4 µg/mL (cephalexin) to 1 µg/mL (cephalexin-loaded niosome) against E. coli and S. aureus, respectively. The findings of our study show that the improvement of cephalexin bioavailability and prolonged drug release profile could be obtained by niosomal formulation as a favorable antibiotic drug delivery system.