Harmful non-indigenous species (NIS) impose great economic and environmental impacts globally, but little is known about their impacts in Southeast Asia. Lack of knowledge of the magnitude of the problem hinders the allocation of appropriate resources for NIS prevention and management. We used benefit-cost analysis embedded in a Monte-Carlo simulation model and analysed economic and environmental impacts of NIS in the region to estimate the total burden of NIS in Southeast Asia. The total annual loss caused by NIS to agriculture, human health and the environment in Southeast Asia is estimated to be US$33.5 billion (5th and 95th percentile US$25.8–39.8 billion). Losses and costs to the agricultural sector are estimated to be nearly 90% of the total (US$23.4–33.9 billion), while the annual costs associated with human health and the environment are US$1.85 billion (US$1.4–2.5 billion) and US$2.1 billion (US$0.9–3.3 billion), respectively, although these estimates are based on conservative assumptions. We demonstrate that the economic and environmental impacts of NIS in low and middle-income regions can be considerable and that further measures, such as the adoption of regional risk assessment protocols to inform decisions on prevention and control of NIS in Southeast Asia, could be beneficial.