Prioritizing brownfields for redevelopment in real estate portfolios can contribute to more sustainable regeneration and land management. Owners of large real estate and brownfield portfolios are challenged to allocate their limited resources to the development of the most critical or promising sites, in terms of time and cost efficiency. Authorities worried about the negative impacts of brownfields - in particular in the case of potential contamination - on the environment and society also need to prioritize their resources to those brownfields that most urgently deserve attention and intervention. Yet, numerous factors have to be considered for prioritizing actions, in particular when adhering to sustainability principles. Several multiple-criteria decision analysis (MCDA) approaches and tools have been suggested in order to support these actors in managing their brownfield portfolios. Based on lessons learned from the literature on success factors, sustainability assessment and MCDA approaches, researchers from a recent EU project have developed the web-based Timbre Brownfield Prioritization Tool (TBPT). It facilitates assessment and prioritization of a portfolio of sites on the basis of the probability of successful and sustainable regeneration or according to individually specified objectives. This paper introduces the challenges of brownfield portfolio management in general and reports about the application of the TBPT in five cases: practical test-uses by two large institutional land owners from Germany, a local and a regional administrative body from the Czech Republic, and an expert from a national environmental authority from Romania. Based on literature requirements for sustainability assessment tools and on the end-users' feedbacks from the practical tests, we discuss the TBPT's strengths and weaknesses in order to inform and give recommendations for future development of prioritization tools.