Due to the inter-dependent network of organisms, a microbe’s interaction with the physical environment and its continuous evolution through mutation and horizontal gene transfer, microbial diversity is never static, which makes an analytical approach almost impossible for assessing the risk of the environmental use of microbes. One possible alternative approach could build on concepts developed by the OECD in the early 1990s: familiarity and substantial equivalence. According to the Cartagena Protocol on Biosafety, “the objective of a risk assessment is to identify and evaluate the potential adverse effects of living modified organisms on the conservation and sustainable use of biological diversity in the likely potential receiving environment, taking also into account risks to human health”. The “potential adverse effects” are not always easy to identify and interpreting them in different circumstances has been a long-standing question. Ambiguity surrounding this key word appears to have caused regulatory uncertainty. This chapter addresses the target of risk assessment of environmental application of microbes and the difficulty of using an analytical approach in assessing the risk of micro-organisms used in the environment.