Only a small subset of the hundreds of proteins encoded by the poxvirus genome have been shown to be effective as vaccine and/or therapeutic targets. One of these proteins is A33. Here we assess and dissect the ability of an anti-A33 humanized monoclonal antibody, c6C, to affect vaccinia virus infection in vitro. Enveloped virions (EV) released from infected cells can be sensitive or resistant to neutralization by c6C indicating there are different types of EV particles, extracellular enveloped virions (EEV) and released cellular-associated virions (rCEV), that are biologically distinct. Through a combination of plaque phenotype, confocal imaging, and neutralization assays, we found that c6C differentially affects EV from two different virus strains, IHD-J and WR. Evidence for an anti-A33 resistant EV particle, and strain differences in this phenotype, provides a logical answer as to why certain functional assays in the literature have been unable to detect anti-viral effects of anti-A33 antibodies.