Pseudomonas aeruginosa is very important pathogen causing infections in patients being stricken with burns, cystic fibrosis. Because of their biofilm formations on tissues and insertable tools such as catheters, implants, etc., it is hard to eradicate these bacteria in the health-care environment. In order to decrease infections associated with biofilm infections, new antibiofilm compunds and antibiofilm surfaces for medical area/tools are urgent requirements. For this purpose, available methods require expensive instruments, trained people as well as good laboratory facilities. In this study, a fluorescence-based method was developed for the real-time minimal inhibitor concentration (RT-MIC) assay of Pseudomonas aeruginosa and it was also used as a simple, continuous screening method for detection of antibiofilm activities. By using this method, biofilms could be directly investigated against new antibiofilm agents/surfaces in a continuous mode, unlike the end-point detection methods. We believe it makes a significant contribution for continuous screening of biofilms against antimicrobial compounds/surfaces and analysis of multiple samples in clinical applications.