Introduction In the present study, confocal microscopy, a miniflow cell system, and image analysis were combined to test in situ the effect of antimicrobials and alkali on biofilms of Enterococcus faecalis, Lactobacillus paracasei, Streptococcus anginosus, and Streptococcus gordonii isolated from root canals with persistent infections. Methods Biofilms formed for 24 hours were exposed for 5 minutes to alkali (pH = 12), chlorhexidine digluconate (2.5%), EDTA (50 mmol/L), and sodium hypochlorite (1%). The biofilms were then characterized by using fluorescent markers targeting cell membrane integrity (LIVE/DEAD) and metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride and fluorescein diacetate). In addition, the biofilm architecture and the extent to which coating of the substrate surface with collagen influenced the resistance pattern to the chemicals were also analyzed. Results NaOCl (1%) affected the membrane integrity of all organisms and removed most biofilm cells. Exposure to EDTA (50 mmol/L) affected the membrane integrity in all organisms but failed to remove more than a few cells in biofilms of E. faecalis, L. paracasei, and S. anginosus. Chlorhexidine (2.5%) had a mild effect on the membrane integrity of E. faecalis and removed only 50% of its biofilm cells The effects were substratum-dependent, and most organisms displayed increased resistance to the antimicrobials on collagen-coated surfaces. Conclusions The biofilm system developed here was sensitive and differences in cell membrane integrity and removal of biofilm cells after exposure to antimicrobials commonly used in endodontics was discernible.