The majority of eukaryotes encode some intron-containing pre-tRNAs. Splicing of these pre-tRNAs requires a dedicated tRNA splicing machinery. The fungal and trypanosome tRNA ligase, Trl1, and the human RNA ligase, RTCB, catalyze an essential step in tRNA splicing. However, Trl1 and RTCB are nonhomologous and biochemically and structurally distinct from each other. Therefore, Trl1 could serve as a broad-spectrum antifungal and anti-trypanosomal target. While the functions and requirements of the three catalytic Trl1 domains have been extensively characterized in the model yeast Saccharomyces cerevisiae, the roles of Trl1 orthologs in pathogenic fungi remain unexplored. Here, we validate Trl1 as one of the few promising novel drug targets for the development of antifungal therapeutics. Functional analyses of the three Trl1 domains show that only the “sealing” domain is essential for growth and viability in Candida albicans and Aspergillus fumigatus. In contrast, the two “healing” domains are dispensable in these pathogenic fungi, suggesting the presence of redundant healing enzymes, unlike in S. cerevisiae. These findings indicate that only the sealing domain is a good drug target. Our analysis also shows that the Mucor enzyme, which only contains the sealing domain, is essential. Using a Caenorhabditis elegans infection model of C. albicans, we further demonstrated that inhibiting Trl1 expression protects worms during an established infection. In contrast to these fungal pathogens, we show that all three domains of Trl1 are essential in Trypanosoma brucei. Our findings show that the essentiality of the Trl1 sealing is conserved in important human pathogens and provides an impetus for future drug development.