Abstract: In the present study, the effects of combined metabolites produced by lactic acid bacteria were tested on food pathogenic and contaminant microorganisms. Totally, eight strains of Lactobacillus acidophilus (FTDC 2804, FTDC 0785, FTDC 8592, FTDC 1295, FTDC 4793, FTDC 4462, FTDC 0582 and FTDC 2916) were used which cultivated in four types of agro waste substrates including pineapple waste, soy whey, cabbage and molasses. The inhibitory effects of L. acidophilus metabolites (lactic acid, hydrogen peroxide, acetaldehyde, diacetyl and bacteriocin) were determined by agar well diffusion method on two pathogenic bacteria; Staphylococcus aureus and Escherichia coli. Metabolites of different L. acidophilus strains cultured in different agro waste substrates showed diverse inhibitory effects. Among all, the highest inhibition zone was obtained with the strains cultivated in pineapple waste, such as, L. acidophilus FTDC 4462 strain (10.57±0.10 mm for S. aureus and 11.13±0.45 mm for E. coli). It can be concluded that L. acidophilus species has the ability to grow in agro waste materials and produce beneficial metabolites with antibacterial activities.