Exploring the antibacterial potential of neem oil (Azadirachta indica) in combination with gentamicin (GEN) against pathogenic molds, especially Pseudomonas aeruginosa, has drawn concern due to the quest for natural treatment options against incurable diseases. Prospective research directions include looking for natural cures for many of the currently incurable diseases available now. microbial identification system, were used to identify the isolates. The research utilized a range of methods, such as the diffusion agar well (AWD) assays, TEM (transmission electron microscopy) analysis, minimum inhibitory concentration (MIC) assays, and real-time PCR (RT-qPCR) to analyze bacterial expression and the antibacterial action of neem oil (Azadirachta indica) combined with gentamicin (GEN) against the pathogenic bacteria Pseudomonas aeruginosa between others. The combined effects of neem oil and GEN on P. aeruginosa are extensively investigated in this study, with particular attention paid to the downregulation of the virulent factor gene phzM, inhibition of biofilm formation, morphological changes, and gene expression, in that proportion. Results show that neem oil has strong antibacterial activity against P. aeruginosa and S. aureus, preventing the formation of biofilms and causing morphological alterations. In addition, the synergistic effects of neem oil and GEN are demonstrated, together in order with the downregulation of the phzM gene in P. aeruginosa. These results highlight the increased effectiveness of neem oil when combined with GEN and point to the potential of the oil as a stand-alone antibacterial agent. They also highlight the need for more study to overcome antibiotic resistance in harmful bacteria. Overall, our results point to a possibility that neem oil extract, either alone or in combination with GEN, can suppress the development of pathogenic microbes. This synergistic impact is related to the downregulation of a virulence factor gene. To address the drug resistance linked to pathogenic microbes, greater study is required. Novelty of study addresses the important issue of antibiotic resistance by exploring the potential synergistic effects of neem oil and gentamicin on pathogenic bacteria, particularly Pseudomonas aeruginosa. This topic is highly relevant in the context of the global challenge of antimicrobial resistance. Novelty and relevance the important issue of antibiotic resistance by exploring the potential synergistic effects of neem oil and gentamicin on pathogenic bacteria, particularly Pseudomonas aeruginosa. This topic is highly relevant in the context of the global challenge of antimicrobial resistance.