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Plant mitochondrial genomes (mitogenomes) experience remarkable levels of horizontal gene transfer, including the recent discovery that orchids anciently acquired DNA from fungal mitogenomes. Thus far, however, there is no evidence that any of the genes from this interkingdom horizontal gene transfer are functional in orchid mitogenomes. Here, we applied a specialized sequencing approach to the orchid Corallorhiza maculata and found that some fungal-derived tRNA genes in the transferred region are transcribed, post-transcriptionally modified, and aminoacylated. In contrast, all the transferred protein-coding sequences appear to be pseudogenes. These findings show that fungal horizontal gene transfer has altered the composition of the orchid mitochondrial tRNA pool and suggest that these foreign tRNAs function in translation. The exceptional capacity of tRNAs for horizontal gene transfer and functional replacement is further illustrated by the diversity of tRNA genes in the C. maculata mitogenome, which also include genes of plastid and bacterial origin in addition to their native mitochondrial counterparts.