Ortho-benzyne is a potentially important precursor for polycyclic aromatic hydrocarbon formation, but much is still unknown about its chemistry. In this work, we report on a combined experimental and theoretical study of the o-benzyne + acetylene reaction and employ double imaging threshold photoelectron photoion coincidence spectroscopy to investigate the reaction products with isomer specificity. Based on photoion mass-selected threshold photoelectron spectra, Franck-Condon simulations, and ionization cross section calculations, we conclude that phenylacetylene and benzocyclobutadiene (PA : BCBdiene) are formed at a non-equilibrium ratio of 2 : 1, respectively, in a pyrolysis microreactor at a temperature of 1050 K and a pressure of ∼20 mbar. The C₈H₆ potential energy surface (PES) is explored to rationalize the formation of the reaction products. Previously unidentified pathways have been found by considering the open-shell singlet (OSS) character of various C₈H₆ reactive intermediates. Based on the PES data, a kinetic model is constructed to estimate equilibrium abundances of the two products. New insights into the reaction mechanism-with a focus on the OSS intermediates-and the products formed in the o-benzyne + acetylene reaction provide a greater level of understanding of the o-benzyne reactivity during the formation of aromatic hydrocarbons in combustion environments as well as in outflows of carbon-rich stars.