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When plant biomass is anaerobically digested, seeds may survive the energy production process and contaminate the digestate. Hard-seeded (HS), i.e., physically dormant, species were found to be difficult to inactivate. Here, we aimed to verify this finding from lab-scale experimental reactors (ERs) in a full-scale commercial reactor (CR). In addition, we tested seed survival in a pH-buffered water bath (WB). Seeds were exposed to CR, ER and WB treatments at 42 °C for a maximum of 36 days. The viability of seeds was checked by measuring germination and response to tetrazolium staining and modeled as a function of exposure time using a dose–response approach. CR killed seeds more effectively than ER and WB treatments. The non-HS reference species, Chenopodium album, was completely inactivated by all treatments. Responses of the HS species ranged from complete inactivation to complete insensitivity. The most resistant was Malva sylvestris. The least resistant species were inactivated mainly by temperature, while additional mortality factors were effective in the more resistant species. We concluded that mesophilic AD in CRs can reduce the risk of seed contamination in the digestate for non-HS but not for HS species. Moreover, WB treatments seem suitable to estimate the minimum mortality of non-HS species in CR.