Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that mends topoisomerase 1-mediated DNA damage. Tdp1 is a current inhibition target for the development of improved anticancer treatments, as its inhibition may enhance the therapeutic effect of topoisomerase 1 poisons. Here, we report a study on the development of a novel class of Tdp1 inhibitors that is based on the octahydro-2H-chromene scaffold. Inhibition and binding assays revealed that these compounds are potent inhibitors of Tdp1, with IC50 and KD values in the low micromolar concentration range. Molecular modelling predicted plausible conformations of the active ligands, blocking access to the enzymatic machinery of Tdp1. Our results thus help establish a structural-activity relationship for octahydro-2H-chromene-based Tdp1 inhibitors, which will be useful for future Tdp1 inhibitor development work.