Researchers are working to develop a new class of antimicrobial agents due to the high prevalence of microbial diseases and their multidrug resistance properties. The use of indole with nanoparticles as new antimicrobial agent formulations is a modern and innovative approach to drug development. Current study demonstrates the antimicrobial efficacy of indole with calcium carbonate Nanoparticles (caco3-NPs) against gram negative, opportunistic bacterial infection, Pseudomonas aeruginosa. Caco3-NPs alone show a significant antibacterial activity, with inhibition zones range from 8 to 17 mm at concentration 10.000 μg /ml. While the indole alone too with inhibition zones range from 27 to 35 mm. The minimum inhibitory concentration(MIC) of caco3-NPs showed a strong antibacterial activity 312.5 μg /ml. At the same time, the MIC of Indole was 156.2 μg /ml. When combined Indole with caco3-NPs, they show increased inhibition rate of growth and biofilm formation by persister cell of P. aeruginosa. Caco3-NPs greatly aided biofilm inhibition with Indole about 63.32 percent at 10 mg /ml concentration. As a result, caco3-NPs and Indole significantly impacted P. aeruginosa persister cells' biofilms formation.