We have previously described several different chemical series of bicyclic prolyl oligopeptidase (POP) inhibitors as probes for neurodegenerative diseases that demonstrated nanomolar activity in vitro and submicromolar activity in cellulo. The more recent implication of POP in cancer, together with homologous fibroblast activation protein α (FAP), implicated in tumor growth, led us to consider developing POP/FAP dual inhibitors as a promising strategy for the development of cancer therapeutics. At this stage, we thought to evaluate the requirements for selectivity of inhibitors for POP over FAP and to evaluate molecular platforms that would enable the development of selective POP and dual POP/FAP inhibitors. We report herein docking-guided design of a new bicyclic scaffold and synthesis of both covalent and non-covalent bicyclic inhibitors. Biological evaluation of first-of-their-kind [4.3.0] bicyclic compounds confirmed that reactive groups, or covalent warheads, are required for inhibitor activity. This work ultimately led to one scaffold yielding new POP-selective inhibitors and a dual inhibitor equipotent to the only drug targeting FAP and POP that ever reached clinical trials.