<p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Background:</span></strong><span style="font-size:10.0pt"> To develop a probiotic formulation for poultry feed, a few poultry gastrointestinal derived lactic acid bacteria (pGIT-d-LAB) were isolated from chicken intestinal specimens and <em>in vitro</em> experiment was performed to evaluate their efficacy as potential probiotic candidate.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Methods:</span></strong><span style="font-size:10.0pt"> A total of 6 strains of LAB: <em>L</em><em>actobacillus brevis</em> (<em>L. brevis</em>)<em>,</em> <em>L</em><em>actobacillus acidophilus </em>(<em>L. acidophilus</em>)<em>, Lactobacillus casei </em>(<em>L. casei</em>)<em>, Pediococci </em>spp.<em>, Lactobacillus fermentum </em>(<em>L. fermentum</em>)<em> </em>and<em> L</em><em>actobacillus plantarum </em>(<em>L. plantarum</em>)<em> </em>were isolated and cultured for collection of Cell Free Supernatant (CFS). CFS collected was tested against pathogenic bacterial isolated from chicken feces as well as prevalent fungal pathogens, utilizing agar-well diffusion techniques. A preliminary investigation into the susceptibility of the pathogens to diverse antibiotics and antifungal drugs was conducted. Bacterial pathogens exhibiting resistance to a minimum of three classes of antibiotics were subsequently identified for pGIT-d-LAB CFS screening. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Results:</span></strong><span style="font-size:10.0pt"> The observed results revealed that the CFS derived from the isolates exhibited varying degrees of growth inhibition against different pathogens. Among the tested pGIT-d-LAB isolates, <em>L. acidophilus </em>demonstrated the most prominent zone of inhibition, measuring 18 <em>mm</em> against <em>Klebsiella pneumoniae</em> ZTAC 1233. Notably, <em>Citrobacter diversus</em> ZTAC 1255 showed resistance to all tested pGIT-d-LAB. Quantification of the metabolites produced was performed, and peak production levels was determined. <em>L.</em><em> acidophilus </em>produced the highest amount of lactic acid (1.789<em>g/l</em>), <em>Pediococci </em>spp. produced the highest amount of diacetyl and H₂0₂ (1.918<em>g/l</em>) (0.0025<em>g/l</em>) at 48<em> hr</em> peak values respectively. </span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><strong><span style="font-size:10.0pt">Conclusion:</span></strong><span style="font-size:10.0pt"> The test isolates are potential probiotic candidates for controlling pathogens in poultry. </span></span></p>