This review critically updates the Pariza microbial enzyme safety evaluation decision tree, which has been used by industry since the turn of the century, to reflect current biotechnology tools and microbial safety assessment practices. The updated decision tree preserves the principle that enzyme safety depends primarily on the safety of the selected microbial production organism. Our review adds refinements to be more inclusive of production organisms improved by random or targeted mutagenesis. Its questions are redesigned to consider current technologies, such as gene synthesis, gene editing, whole genome sequencing, and safety concepts such as Qualified Presumption of Safety (QPS) status of production organisms. With these updates, the decision tree continues to support the view that responsible genetic improvement poses few, if any, safety concerns. It also provides a robust and globally relevant framework for assessing the safety of food and feed enzymes and may serve as model for other microbially produced ingredients and dietary proteins.