The ability to overcome drug resistance in outbreaks of?Candida albicans?infection is an unmet need in health management. Here, we investigated?CDC8, which encodes thymidylate kinase (TMPK), as a potential drug target for the treatment of?C. albicansinfection. In this study, we found that the specific region spanning amino acids 106-123, namely, the Ca-loop of?C. albicans?TMPK (CaTMPK) contributes to the hyperactivity of this enzyme compared to the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/ deoxy-5-Fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, CaTMPK but not hTMPK enables dUTP/5-FdUTP-mediated DNA toxicity in yeast. CRISPR-mediated deletion of this Ca-loop in?C. albicans?demonstrated the critical role of this Ca-loop in fungal growth and susceptibility to 5-Fluorouridine (5-FUrd). Moreover, pathogenic and drug-resistant?C. albicans?clones were similarly sensitive to 5-FUrd. Thus, this study not only identified a target site for the development of CaTMPK-selective drugs but also revealed 5-FUrd to be a potential drug for the treatment of?C. albicans?infection.