Background and Aim: Probiotics are beneficial microorganisms for humans and animals. In this study, we developed a microencapsulated probiotic with antibacterial activity against avian pathogenic Escherichia coli (APEC). Materials and Methods: Alignment of the 16S rRNA sequences of the isolate WU222001 with those deposited in GenBank revealed that the isolate was Pediococcus acidilactici with 99.6% homology. This bacterium was characterized as a probiotic based on its tolerance toward in vitro gastrointestinal tract (GIT) conditions, hydrophobicity, and auto-aggregation. The antibacterial activity of the probiotic’s culture supernatant against APEC was investigated using a broth microdilution assay. Pediococcus acidilactici was microencapsulated using sodium alginate and agar with diameters ranging from 47 to 61 µm. Then, physicochemical characteristics and stability of the microcapsules were determined. Results: The isolate was characterized as a probiotic based on its resistance to low pH, bile salts, and pancreatin, with relative values of 79.2%, 70.95%, and 90.64%, respectively. Furthermore, the bacterium exhibited 79.56% auto-aggregation and 55.25% hydrophobicity at 24 h. The probiotic’s culture supernatant exhibited strong antibacterial activity against clinical APEC isolates with minimum inhibitory concentration and minimum bactericidal concentration of 12.5% and 25% v/v, respectively. Microencapsulation-enhanced bacterial viability in GIT compared to free cells. Moreover, 89.65% of the encapsulated cells were released into the simulated intestinal fluid within 4 h. The viable count in microcapsules was 63.19% after 3 months of storage at 4°C. Conclusion: The results indicated that the culture supernatant of P. acidilactici inhibited the growth of APEC. In addition, microencapsulation extends the viability of P. acidilactici under harsh conditions, indicating its potential application in the feed production. Keywords: antibacterial activity, avian pathogenic Escherichia coli, microcapsule, microencapsulation, Pediococcus acidilactici, probiotics.