This paper evaluated the use of soft-probe scanning electrochemical microscopy complementarily with confocal laser scanning microscopy to study the effects of different antimicrobial agents and treatments on E. coli DH5α biofilm. The antimicrobial agents were sodium azide, silver nanoparticles, and a flashlight. The effects of these agents were monitored by measuring the change in biofilm properties, such as biofilm biomass, live/dead studies, and surface activity. The results showed that sodium azide, silver nanoparticles, and the flashlight effectively killed E. coli biofilms and explained the mode of action for each treatment. Sodium azide was more effective in killing the biofilm after a short treatment time by blocking the ATPase, while silver nanoparticles were more effective at killing the biofilm after longer treatment times through several antibiofilm actions. This work showed that scanning electrochemical microscopy (SECM) is a very valuable tool for studying the effects of antimicrobial agents on biofilms. SECM is a sensitive technique that can be used to monitor the changes in biofilm properties in real-time. Additionally, SECM does not require any sample preparation, which makes it a convenient and efficient technique. Overall, the results of this study could be used to develop new strategies for treating E. coli biofilm infections and provide valuable insights into the use of SECM to study the effects of antimicrobial agents on E. coli biofilms.