Piperacillin (Pip) is a broad spectrum beta-lactam against most Gram-positive and Gram-negative aerobic and anaerobic bacteria. However, bacterial resistance restricts its benefits for the treatment of infectious diseases. Recently, nanoliposomal systems have been investigated as encouraging strategies to address this issue owing to their immense potential. We aimed to encapsulate Pip in liposomal nanoparticles and study their antibacterial activities in vitro against Pseudomonas aeruginosa (P. aeruginosa). Different liposomes were prepared based on the freeze-drying of a monophase solution method. Then, they were characterized in terms of size, zeta potential, polydispersity-index, and morphology. For further analysis, spectra of ATR-FTIR and XRD were taken for liposomal Pip. Encapsulation efficiency (EE) was determined via agar diffusion assay. Also, minimum inhibitory concentrations (MICs) were investigated by the standard broth macro-dilution method. The liposomes were from 100.9 to 444.13 nm with z-potential of - 30.70 to - 10.57 mV. EE of the selected formulation was 53.1%. TEM results showed that the liposomes were nanosized and almost spherical. ATR-FTIR results confirmed the full encapsulation of Pip in nanoliposomes. The X-ray pattern indicated that the liposomal Pip was amorphous. The MIC (10.6 mu g/ml) of the nanoliposomal Pip against P. aeruginosa was one-half of the MIC (21.25 mu g/ml) of free Pip for the same organisms. Considering four aspects (nanosized liposomes, no need for sterilization, suitable EE and enhanced antibacterial effects), this preparation method seems promising and may be used to overcome the bacterial resistance relative to Pip.