Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2′,3′-cyclic-PO4 and 5′-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3′-OH, 2′-PO4, and 5′-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus. The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2′,3′-cyclic-PO4 and 5′-OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.