Zoonoses incidences continue to play significant roles in both human and veterinary medicine and their eradication especially in the developing country like Nigeria, is difficult. Zoonotic food-borne infections caused by Gram-positive and Gram-negative bacteria are re-emerging. Several findings indicated that the cell-free supernatant derived from some Lactic Acid Bacteria have some antibacterial effects when applied directly or added to food during packaging. The aim of this study was to investigate the In vitro antagonistic effect of cell-free supernatant derived from some LAB species isolated from whey and Nunu (obtained from a locally fermented milk product) against some zoonotic bacteria pathogens (Escherichia coli, Salmonella sp. and Staphylococcus aureus. Streptococcus sp. and Salmonella sp. Four (4) LAB isolates were isolated, identified using basic morphological and biochemical characterization and screened on the basis of functional and technological characterization. The Cell-free supernatant from these strains, Lactobacillus casei LB01, Lactobacillus sp. LB02, Lactobacillus acidophilus LB03 and Lactobacillus plantarum LB04 were prepared by centrifugation at 10,000 rpm for 5 minutes and used against the indicator organisms at 10⁷ CFU/ml using agar well diffusion method. The cell-free supernatant inhibited the growth of both Gram- positive and Gram-negative bacteria strains of veterinary origins. Our findings suggest that the LAB strains have the potential for antibiotic alternatives for the control of several zoonotic diseases. This holds a great advantage in the current efforts to tackling and reducing the prevalence and spread of zoonotic diseases which are re-emerging in Nigeria.