Arboviruses are not routinely screened in blood banks in Colombia. Therefore, collecting blood from asymptomatic infected blood donors with high viremias is a risk in blood banks located in endemic regions. In addition, the prevalence of arbovirosis in the general population changes across epidemiological periods, which is also expectable in blood donors. This study aimed to estimate and compare the prevalence of arbovirus infections in blood donors in Colombia between an epidemic and endemic period. An analytical cross-sectional study compared the prevalence of DENV, ZIKV, and CHIKV infections in blood donors in Colombia between an epidemic (November 2019 to February 2020, n = 462) and an endemic period (November 2021 to August 2022, n = 1119). Viral RNA was purified from the sera of accepted blood donors in four blood banks in endemic regions (0–1984 masl) and two in non-endemic regions (2200–2600 masl). DENV, ZIKV, and CHIKV RNA were detected using a previously standardized multiplex semi-nested RT-PCR protocol. Sample processing was conducted in fresh samples (maximum seven days post-donation). Prevalences are presented as percentages, and comparisons between epidemic and endemic periods were made using the chi-square or Fisher's exact test, with Bonferroni's test for multiple comparisons (p < .05; Stata v16). The total prevalence of DENV, ZIKV, and CHIKV infections in blood donors was 29.7% in endemic and 19.8% in non-endemic regions during the epidemic period (2019–2020). In contrast, the prevalence significantly decreased to 4.9% in endemic and 5.6% in non-endemic regions during the endemic period (2021–2022; p < .05; Figure A). FIGURE A. Prevalence of arbovirus infections in blood donors from endemic and non-endemic regions of Colombia during two epidemiological periods.