Background: Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related members of the Semliki Forest complex within the genus alphavirus and are transmitted by arthropods, causing acute febrile illness in humans. CHIKV has spread to almost all continents, whereas autochthonous MAYV infections have been reported in South America and in the Caribbean. Nevertheless, there was concern about potential spread of MAYV to other regions similar to CHIKV in the past. The risk for transmission of emerging viruses by blood transfusion and the safety of plasma-derived medicinal products (PDMPs) are constant concerns. The manufacturing processes of PDMPs include procedures to inactivate/remove viruses. Methods: In this study, we investigated the reduction of MAYV and CHIKV by heat inactivation in various matrices, solvent/detergent treatment and nanofiltration. Results: Unexpectedly, MAYV was significantly more resistant to heat and solvent/detergent treatment compared to CHIKV. However, being similar in size, both MAYV and CHIKV were removed below the detection limit by 35 nm virus filters. Conclusions: The inactivation profiles of different alphavirus members vary considerably, even within the Semliki Forest Complex. However, robust dedicated viral inactivation/removal procedures commonly used in the plasma product industry are effective in inactivating or removing MAYV and CHIKV.