Abstract <p>Carbon nanotubes (CNTs) emerged as nanomaterials with a wide variety of applications, e.g., as boosters of bioprocesses efficiency. The stimulation of the production of the blue pigment called pyocyanin is one of numerous examples. Moreover, its importance comes from the potential anticancer properties of the pigment. Therefore, this contribution evaluated different commercially available multi-walled carbon nanotubes (MWCNTs) in pyocyanin production using the Design of Experiment methodology. The interactions between pigment-producing bacteria and nanomaterials were revealed as well. Moreover, the purified pigment was tested against normal and cancer cell lines. Interestingly, the results showed that all tested CNTs stimulated pyocyanin production. The most effective CNTs were used in the process optimisation in terms of temperature (32&#xa0;°C) and carbon nanomaterial concentration (812&#xa0;μg/mL). It was also revealed that the optical density and viability of the bacterial culture were elevated, while the pyoverdine production was decreased. Furthermore, no oxidative stress was detected. Moreover, the confocal microscopy study indicated that the cells surrounded the aggregates of MWCNT and produced more proteins within the biofilm structure, compared to the control experiment. The tests on neoplastic cell lines showed an excellent antiproliferative activity of pyocyanin against melanoma without pronounced adverse effects on normal fibroblasts. The nanomaterial incorporated in the bioprocess was successfully reused, making the method sustainable and cost-effective.</p> Key points <p>•&#xa0;<i>The stimulative effect of nanomaterial on pyocyanin production was optimised</i></p> <p>•&#xa0;<i>Nanomaterial can be reused in the bioprocess without losing the stimulative effect</i></p> <p>•&#xa0;<i>Pyocyanin exhibits significant antiproliferative action against melanoma</i></p> Graphical abstract <p></p>