Antimicrobial peptides (AMPs) have become a key solution for controlling multi-drug-resistant (MDR) pathogens, and the nanoencapsulation of AMPs has been used as a strategy to overcome challenges, such as poor stability, adverse interactions, and toxicity. In previous studies, we have shown the potent antimicrobial activity of Octominin against and This study is focused on the nanoencapsulation of Octominin with chitosan (CS) and carboxymethyl chitosan (CMC) as a drug delivery system using the ionotropic gelation technique. Octominin-encapsulated CS nanoparticles (Octominin-CNPs) had an average diameter and zeta potential of 372.80 ± 2.31 nm and +51.23 ± 0.38 mV, respectively, while encapsulation efficiency and loading capacity were 96.49 and 40.20%, respectively. Furthermore, Octominin-CNPs showed an initial rapid and later sustained biphasic release profile, and up to 88.26 ± 3.26% of the total Octominin release until 96 h. Transmission electron microscopy data showed the irregular shape of the Octominin-CNPs with aggregations. In vitro and in vivo toxicity of Octominin-CNPs was significantly lower than the Octominin at higher concentrations. The antifungal and antibacterial activities of Octominin-CNPs were slightly higher than those of Octominin in both the time-kill kinetic and microbial viability assays against and , respectively. Mode of action assessments of Octominin-CNPs revealed that morphological alterations, cell membrane permeability alterations, and reactive oxygen species generation were slightly higher than those of Octominin at the tested concentrations against both and . In antibiofilm activity assays, Octominin-CNPs showed slightly higher biofilm inhibition and biofilm eradication activities compared to that of Octominin. In conclusion, Octominin was successfully encapsulated into CS, and Octominin-CNPs showed lower toxicity and greater antimicrobial activity against and compared to Octominin.