Antibiotic resistance is one of the biggest threats to global health resulting in an increasing number of people suffering from severe illnesses or dying due to infections that were once easily curable with antibiotics.Pseudomonas aeruginosais a major pathogen that has rapidly developed antibiotic resistance and WHO has categorised this pathogen under the critical list. DNA aptamers can act as a potential candidate for novel antimicrobial agents. In this study, we demonstrated that an existing aptamer is able to affect the growth ofP. aeruginosa.A computational screen for aptamers that could bind to a well-conserved and essential outer membrane protein, BamA in Gram-negative bacteria was conducted. Molecular docking of about 100 functional DNA aptamers with BamA protein was performed via both local and global docking approaches. Additionally, genetic algorithm analysis was carried out to rank the aptamers based on their binding affinity. The top hits of aptamers with good binding to BamA protein were synthesised to investigate their in vitro antibacterial activity. Among all aptamers, Apt31, which is known to bind to an antitumor, Daunomycin, exhibited the highest HADDOCK score and resulted in a significant (p < 0.05) reduction inP. aeruginosagrowth. Apt31 also induced membrane disruption that resulted in DNA leakage. Hence, computational screening may result in the identification of aptamers that bind to the desired active site with high affinity.