Introduction: Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, poses a significant threat in hospital settings, causing nosocomial infections with severe consequences. The bacterium’s high antibiotic resistance, particularly through biofilm formation via quorum sensing (QS), complicates treatment strategies. This study explores the potential of eucalyptus (Melaleuca leucadendra), known for its antimicrobial properties especially due to 1,8-cineole, ecoenzyme in inhibiting Pseudomonas aeruginosa. Materials and Methods: This study is a posttest-only control group design. Pseudomonas aeruginosa bacteria were samped using simple random sampling. The study utilized the agar-well diffusion method on Mueller Hinton agar to assess the antibacterial effect of eucalyptus (Melaleuca leucadendra) ecoenzyme. Concentrations tested ranged from 10% to 100% with three repetitions and incubation at 37 °C for 24 hours. Data were obtained by measuring the inhibition zone using calipers. Results: Contrary to expectations, the study found no antibacterial effect of eucalyptus (Melaleuca leucadendra) ecoenzyme on Pseudomonas aeruginosa at all concentrations tested (100%, 90%, 70%, 60%, 50%, 40%, 30%, 20%, and 10%). Conclusion: The investigation into eucalyptus (Melaleuca leucadendra) ecoenzyme revealed no discernible antibacterial activity against Pseudomonas aeruginosa. These findings challenge the initial hypothesis and underscore the importance of thorough experimentation in assessing the potential of natural agents for combating nosocomial infections. Further research is needed to elucidate the complex interactions between Pseudomonas aeruginosa and eucalyputs (Melaleuca leucadendra).