Inhibitory cystine knot (ICK) peptides in spider venom are valuable bioactive peptides due to their therapeutic potential in a wide range of diseases, including chronic pain and epilepsy. Phlo3a which belongs to family 1 spider toxins (NaSpTx1) is a newly discovered 33 residue long Australian spider-venom ICK peptide and inhibits Nav channels involved in pain (hNav1.7, hNav1.3) and epilepsy (hNav1.2). Recombinantly expressed Phlo3a potently blocked these channels without affecting the voltage-dependence of activation, steady-state inactivation or recovery from inactivation, reminiscent of NaSpTx1 toxins HwTx-IV and HnTx-III that trap the domain II voltage sensor in the closed state. Using site-directed mutagenesis and recombinant expression in E. coli, we produced 50+ Phlo3a analogues to define its functional surface and optimize its potency and subtype selectivity. These analogues were designed based on an Ala scan, sequence conservation, and existing structure-function studies of NaSpTx1 peptides. The purified analogues were tested over hNav1.2, hNav1.3, and hNav1.7 in an automated whole-cell patch clamp electrophysiology system. We successfully produced analogues which improved the potency and selectivity at hNav1.2 or improved the selectivity at hNav1.7 depending on the nature and position of the mutation. Based on these data, we designed single and double mutations that enhanced the potency at these targets a further 3-fold and/or improved subtype selectivity. In conclusion, bioactivity data on Phlo3a analogues at three hNav channels has provided valuable new insight into the structure-function of spider-venom peptide inhibitors at hNav1.2, hNav1.3, and hNav1.7.