Microbial biofilms demonstrate an astounding ability to withstand stress, extremes of temperature, drug treatment and other harsh conditions imposed by the surrounding environment. When biofilms are exposed to antimicrobials, most of the cells can be killed, though leaving a subpopulation of biofilm persisters unaffected. The three-dimensional structure of biofilms serves as a protective barrier against the immune system and prevents persisters within the biofilms from being eradicated by immune cells. Biofilms demonstrate extensive structural, chemical and biological heterogeneity, containing cells in various physiological states. Treatment of C. albicans biofilms with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamate (DDTC) led to higher levels of endogenous ROS and an 18- to 200-fold reduction of the miconazole-tolerant persister fraction. The existence of persister cells in a wide variety of microbial subpopulations is becoming increasingly evident from recent studies. Persister cells pose an important threat to healthcare because they are able to withstand the current therapeutic regimens.