DNA interstrand crosslinks (ICLs) are toxic lesions that can block essential biological processes. Here we show Trypanosoma cruzi, the causative agent of Chagas disease, is susceptible to ICL-inducing compounds including mechlorethamine and novel nitroreductase-activated prodrugs that have potential in treating this infection. To resolve such lesions, cells co-opt enzymes from "classical" DNA repair pathways that alongside dedicated factors operate in replication-dependent and -independent mechanisms. To assess ICL repair in T. cruzi, orthologues of SNM1, MRE11 and CSB were identified and their function assessed. The T. cruzi enzymes could complement the mechlorethamine susceptibility phenotype displayed by corresponding yeast and/or T. brucei null confirming their role as ICL repair factors while GFP-tagged TcSNM1, TcMRE11 and TcCSB were shown to localise to the nuclei of insect and/or intracellular form parasites. Gene disruption demonstrated that while each activity was non-essential for T. cruzi viability, nulls displayed a growth defect in at least one life cycle stage with TcMRE11-deficient trypomastigotes also compromised in mammalian cell infectivity. Phenotyping revealed all nulls were more susceptible to mechlorethamine than controls, a trait complemented by re-expression of the deleted gene. To assess interplay, the gene disruption approach was extended to generate T. cruzi deficient in TcSNM1/TcMRE11 or in TcSNM1/TcCSB. Analysis demonstrated these activities functioned across two ICL repair pathways with TcSNM1 and TcMRE11 postulated to operate in a replication-dependent system while TcCSB helps resolve transcription-blocking lesions. By unravelling how T. cruzi repairs ICL damage, specific inhibitors targeting repair components could be developed and used to increase the potency of trypanocidal ICL-inducing compounds.