Puerto Rico (PR) has a long history of vector-borne disease and insecticide-resistant Aedes aegypti (L.). Defining contributing mechanisms behind phenotypic resistance is critical for effective vector control intervention. However, previous studies from PR have each focused on only one mechanism of pyrethroid resistance. This study examines the contribution of P450-mediated enzymatic detoxification and sodium channel target site changes to the overall resistance phenotype of Ae. aegypti collected from San Juan, PR, in 2012. Screening of a panel of toxicants found broad resistance relative to the lab susceptible Orlando (ORL1952) strain. We identified significant resistance to representative Type I, Type II, and nonester pyrethroids, a sodium channel blocker, and a sodium channel blocking inhibitor, all of which interact with the sodium channel. Testing of fipronil, a chloride channel agonist, also showed low but significant levels of resistance. In contrast, the PR and ORL1952 strains were equally susceptible to chlorfenapyr, which has been suggested as an alternative public health insecticide. Molecular characterization of the strain indicated that two common sodium channel mutations were fixed in the population. Topical bioassay with piperonyl butoxide (PBO) indicated cytochrome P450-mediated detoxification accounts for approximately half of the resistance profile. Transcript expression screening of cytochrome P450s and glutathione-S-transferases identified the presence of overexpressed transcripts. This study of Puerto Rican Ae. aegypti with significant contributions from both genetic changes and enzymatic detoxification highlights the necessity of monitoring for resistance but also defining the multiple resistance mechanisms to inform effective mosquito control.