Disinfection is an important strategy to control the environmental transmission of human viruses. Specifically, UV254 is increasingly used to disinfect from clinical items or surfaces to drinking water. RNA virus populations have high mutation rates and large population numbers that allow them to rapidly adapt to stressors. It is therefore likely that variants with enhanced resistance to disinfection may eventually emerge in a virus population. The genetic basis and mechanisms that underpin the emergence of virus resistance to disinfection remain, however, unknown. The goal of this study was to investigate the emergence of echoviruses resistant to UV254 disinfection in vitro, and to characterize the genotypic and phenotypic changes in the resistant populations as compared to the wild-type. Resistant virus populations emerged in mutation-accumulation laboratory experiments. Each experiment consisted in exposing echovirus suspensions to a UV254 fluence such that a 4.5 log10 reduction in infective viruses was achieved, and then regrowing the survivors by infecting BGM cells at a very low multiplicity of infection (MOI>0.001). Unexposed control experiments were conducted in parallel by diluting the virus suspensions instead of exposing them to UV254 prior to regrowth. We demonstrated that echovirus populations became increasingly resistant to UV254. Interestingly, a similar extent of resistance was observed in the unexposed control experiments. Fixed mutations were repeatedly identified by next generation sequencing in the 2C and 3D protein region, both involved in the genome replication. The wild-type and resistant echovirus populations exhibited an equivalent replicative fitness under standard culturing conditions. However, the UV254-resistant mutants were more resistant to ribavirin and guanidine hydrochloride. Finally, the exposed populations were able to maintain the production of infective progeny despite the effect of such chemical mutagens. Combined, these findings suggest that multi-resistance of echovirus to UV254 disinfection and treatment with mutagenic nucleotide analogs may arise in virus populations that, subjected to repeated dramatic bottlenecks, evolve towards an increased replication fidelity. The outcomes of this work show that enteric human pathogens may exhibit multi-resistance to disinfection and clinical antiviral therapies. Ultimately, the results from this work may impact future strategies to ensure adequate control of virus environmental transmission.