Bacterial infections cause severe losses in poultry farming. Clostridium perfringens and Escherichia- coli are two major pathogenic bacteria readily found in the broiler environment. They cause high- morbidity and mortality in poultry worldwide as a result of necrotic enteritis and colibacillosis, respectively. Furthermore, Staphylococcus aureus is an important cause of omphalitis, bumble foot and gangrenous dermatitis. Antimicrobial resistance is one of the most serious global public health threats that necessitates alternative strategies. Probiotics have been emerging as a safe and effective alternative to antibiotics. Lactobacillus being the most commonly used probiotic. Therefore, here the antimicrobial activity of Lactobacillus acidophilus against some pathogens namely Clostridium perfringens, Staphylococcus aureus and Escherichia coli were tested. The antibacterial effect of L. acidophilus was investigated by time kill assay, agar well diffusion and agar spot test. The obtained results showed that L. acidophilus had strong antimicrobial activity against the three bacterial pathogens. Moreover, L. acidophilus exhibited a strong autoaggregating phenotype and marked coaggregation with Cl. perfringens. In addition, inhibition of gas production from Cl. perfringens by L. acidophilus was evaluated. Alpha-toxin has been implicated as one of the major virulence factors of Cl. perfringens inducing avian necrotic enteritis. To investigate whether alpha toxin have adverse effects on L. acidophilus, viability assay proved that L. acidophilus was not remarkably affected by incubation with different concentrations of alpha toxin and different incubation time. Our results suggest that L. acidophilus exhibits strong inhibitory effects against Cl. perfringens, E. coli and S. aureus, and has strong co-aggregation abilities