Understanding the evolution of microbial diversity is an important and current problem in evolutionary ecology. In this paper, we investigated the role of two established biochemical trade-offs in microbial diversification using a model that connects ecological and evolutionary processes with fundamental aspects of biochemistry. The trade-offs that we investigated are as follows:(1) a trade-off between the rate and affinity of substrate transport; and (2) a trade-off between the rate and yield of ATP production. Our model shows that these biochemical trade-offs can drive evolutionary diversification under the simplest possible ecological conditions: a homogeneous environment containing a single limiting resource. We argue that the results of a number of microbial selection experiments are consistent with the predictions of our model.