Achieving substrate-selectivity is a central element of nature’s approach to synthesis; relying on the ability of a catalyst to discriminate, based on small structural changes, which molecules will move forward in a synthesis. This approach can be challenging to duplicate in the laboratory, but can be powerful when realized. In this work, substrate-selective catalysis is leveraged to discriminate between two intermediates that exist in equilibrium, subsequently directing the final cyclization to arrive at either the linear or angular tricyclic core common to subsets of azaphilone natural products. By using a flavin-dependent monooxygenase (FDMO) in sequence with an acyl transferase (AT), the conversion of several orcinaldehyde substrates directly to the corresponding linear tricyclic azaphilones in a single reaction vessel was achieved. Furthermore, mechanistic studies support that a substrate equilibrium together with enzyme substrate-selectivity play an import role in the selectivity of the final cyclization step. A panel of azaphilone natural products and derivatives thereof were synthesized using this strategy.