Post-transplant cyclophosphamide (PTCy) has contributed to the success of haploidentical hematopoietic stem cell transplantation (HSCT) and is also used in transplantation from matched donors. However, limited data on the immune reconstitution after this type of immunosuppression is available. We aimed to evaluate immune reconstitution after HSCT from unrelated donors, comparing anti-thymocyte globulin (ATG) and PTCy. Consecutive patients undergoing HSCT from unrelated donors and receiving either ATG or PTCy were prospectively included. Immune reconstitution analyses were performed by flow cytometry pre-transplant and on days 30, 60, 90, and 180 post-transplant. We included 36 patients, 20 in the ATG group and 16 in the PTCy group. In the early post-transplant period (day [d]+30), the ATG group showed a higher number of total lymphocytes, T, B, and natural killer (NK) cells compared to the PTCy group. However, at d+180, the PTCy group exhibited a higher number of B cells. On d+60 and d+90, the ATG group displayed higher number of NK cells CD56dim compared to the PTCy group, while on d+180, the PTCy group showed higher number of CD56-, CD16+, and, NKG2D+ NK cells. Naive CD4+, transition CD4+, and naive CD8+ T cells on d+60 were identified as risk factors for acute graft-versus-host disease grade 2-4, and a higher count of CD4+ memory cells on d+180 was identified as a risk factor for chronic graft-versus-host disease. In the context of unrelated allogeneic transplantation, immunosuppression with PTCy was associated with later B-, T- and NK-cell reconstitution compared to ATG.