With the advent of wide-field cosmological surveys, we are approaching samples of hundreds of thousands of galaxy clusters. While such large numbers will help reduce statistical uncertainties, the control of systematics in cluster masses becomes ever more crucial. Here we examine the effects of an important source of systematic uncertainty in galaxy-based cluster mass estimation techniques: the presence of significant dynamical substructure. Dynamical substructure manifests as dynamically distinct subgroups in phase-space, indicating an unrelaxed state. This issue affects around a quarter of clusters in a generally selected sample. We employ a set of mock clusters whose masses have been measured homogeneously with commonly-used galaxy-based mass estimation techniques (kinematic, richness, caustic, radial methods). We use these to study how the relation between observationally estimated and true cluster mass depends on the presence of substructure, as identified by various popular diagnostics. We find that the scatter for an ensemble of clusters does not increase dramatically for clusters with dynamical substructure. However, we find a systematic bias for all methods, such that clusters with significant substructure have higher measured masses than their relaxed counterparts. This bias depends on cluster mass: the most massive clusters are largely unaffected by the presence of significant substructure, but masses are significantly overestimated for lower mass clusters, by ∼ 10% at 10 14 and 20% for 10 13.5 . The use of cluster samples with different levels of substructure can therefore bias certain cosmological parameters up to a level comparable to the typical uncertainties in current cosmological studies.