Coxsackievirus B3 (CVB3) is a picornavirus that is responsible for a significant proportion of human myocarditis. No antiviral treatment is currently available for any picornaviral infections. Previously we showed that amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride inhibit the in vitro enzymatic activity of CVB3 RNA polymerase (3D^pol) and proposed a binding site and mechanism of action. Here we measure and compare the biological activity of ten amiloride analogues and undertake their molecular docking using two algorithms, Surflex-Dock^TM and FRED^TM. We show that substituents at the 5-amino and guanidino groups of amiloride individually modulate the inhibitory potency against CVB3 3D^pol and in combination can produce more potent inhibition. A optimisation of CVB3 3D^pol crystal structure, using homology modelling, reflects with the biological activity of amiloride analogues and suggests an initial structure-activity relationship for this class of compounds. This work has produced a model that successfully differentiates between active and inactive amiloride analogues. Our results support the previously predicted binding site for these compounds and provide insights into the interactions required for inhibition of CVB3 3D^pol, thus forming a basis for development of more potent inhibitors for this promising therapeutic target for treatment of myocarditis.