Human herpesvirus 6 (HHV-6) and CMV reactivation were monitored in a cohort of 72 consecutive haematopoietic stem cell transplant (HSCT) patients using RQ-PCR and antigenaemia assay, respectively. The association between acute GVHD (aGVHD) and HHV-6B/CMV was evaluated. We found that on day 100 the cumulative incidence of grades I-IV aGVHD, grades II-IV aGVHD and grades III-IV aGVHD was 55.6, 27.8 and 13.9%, respectively. Multivariate analysis indicated that HHV-6B reactivation was closely correlated with a higher probability of grade II-IV aGVHD by day 30 (Hazard ratio (HR), 8.9; 95% confidence interval (CI), 2.6-31.0; P=0.0006), by day 50 (HR, 6.1; 95% CI, 2.1-17.8; P=0.0010) and by day 100 (HR, 4.8; 95% CI, 1.7-13.6; P=0.0028). However, CMV reactivation did not significantly affect the development of aGVHD by day 50 (HR, 0.8; 95% CI, 0.1-6.7; P=0.8236) and by day 100 (HR, 0.5; 95% CI, 0.1-4.4; P=0.5330) after HSCT. In conclusion, this study demonstrated that active HHV-6B infection, but not CMV, is significantly associated with an increased risk of aGVHD development after HSCT.