Although azole antifungals are considered to be fungistatic, miconazole has fungicidal activity against planktonic Candida albicans cells, presumably associated with the induction of reactive oxygen species (ROS) production. Only few data are available concerning the effect of miconazole against sessile C. albicans cells. In the present study, the fungicidal activity of miconazole against in vitro-grown mature Candida biofilms, and its relationship with the induction of ROS and ROS-dependent apoptosis were examined.The effect of miconazole on mature biofilms formed by 10 C. albicans strains and 5 strains from other Candida species was evaluated by plate counting and measuring the level of ROS induction. MIC tests were performed in the absence and presence of ascorbic acid, a quencher of ROS. The apoptotic population in C. albicans cells was determined using annexin-Cy3.Miconazole showed a significant fungicidal effect against all mature Candida biofilms tested and caused elevated ROS levels, both in planktonic and sessile cells. Addition of ascorbic acid drastically reduced these levels. While ROS quenching decreased the susceptibility to miconazole of planktonic cells of most Candida strains, no reduced fungicidal activity of miconazole against biofilms was observed. Miconazole did not cause a significant increase in apoptosis.ROS levels increased in all Candida biofilms upon addition of miconazole. However, ROS induction was not the only factor that underlies its fungicidal activity, as quenching of ROS did not lead to an enhanced survival of biofilm cells. ROS-induced apoptosis was not observed in C. albicans cells after miconazole treatment.