Infections with the opportunistic yeast Candida glabrata have increased dramatically in recent years. Antifungal therapy of yeast infections commonly employs azoles, such as fluconazole (FLC), but C. glabrata frequently develops resistance to these inhibitors of ergosterol biosynthesis. The pyrimidine analog flucytosine (5-fluorocytosine [5FC]) is highly active versus C. glabrata but is now rarely used clinically due to similar concerns over resistance and, a related concern, the toxicity associated with high doses used to counter resistance. Azole-5FC combination therapy would potentially address these concerns; however, previous studies suggest that 5FC may antagonize azole activity versus C. glabrata. Here, we report that 5FC at subinhibitory concentrations antagonized the activity of FLC 4- to 16-fold versus 8 of 8 C. glabrata isolates tested. 5FC antagonized the activity of other azoles similarly but had only indifferent effects in combination with unrelated antifungals. Since azole resistance in C. glabrata results from transcription factor Pdr1-dependent upregulation of the multidrug transporter gene CDR1, we reasoned that 5FC antagonism might be similarly mediated. Indeed, 5FC-FLC antagonism was abrogated in pdr1Δ and cdr1Δ strains. In further support of this hypothesis, 5FC exposure induced CDR1 expression 6-fold, and this upregulation was Pdr1 dependent. In contrast to azoles, 5FC is not a Cdr1 substrate and so its activation of Pdr1 was unexpected. We observed, however, that 5FC exposure readily induced petite mutants, which exhibit Pdr1-dependent CDR1 upregulation. Thus, mitochondrial dysfunction resulting in Pdr1 activation is the likely basis for 5FC antagonism of azole activity versus C. glabrata.