BackgroundCancer cells release heterogeneous populations of extracellular vesicles (EVs) that transmit aggressive phenotypic traits to recipient cells. We aimed to establish if the heterogenous EVs population or a sub-population is responsible, if we could block undesirable cell-to-cell communication by EVs, and, if some EVs continued to be released, would their undesirable influences on recipient cells continue.MethodsThree triple-negative breast cancer (TNBC) cell lines were used. Non-toxic concentrations of calpeptin, Y27632, manumycin A, GW4869 and combinations thereof were tested to block EVs. Ultracentrifugation-based methods collected EVs, which were then characterised by nanoparticle tracking analysis, immunoblotting, and transmission electron microscopy. A quick screening flow cytometry method evaluated EVs in solution. The influences of EVs on recipient cells’ migration was investigated.ResultsAll EV sub-populations were apparently involved in transmitting undesirable phenotypic characteristics. All compounds/combinations significantly (64–98%) reduced EVs’ release. Our quick screening broadly reflected our more comprehensive EVs analysis. The 2–36% of EVs that continued to be released caused less transmission to recipient cells, but not on a comparable scale to the reduction of EVs release achieved.ConclusionUp to 98% inhibition of EVs’ release was achieved. To prevent the transmission of undesirable phenotypic traits by EVs, their total inhibition may be necessary.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-023-11160-2.