Occurrence of emerging mycotoxins in food and feed is reported worldwide, but studies on their toxic effects are still limited. Our previous study based on in silico approaches has evidenced an association between some cyclic peptidic mycotoxins and non-small cell lung cancer. In this work, an in vitro toxicological investigation of these mycotoxins (enniatin A, enniatin B, beauvericin, apicidin and destruxin B) was performed. Several pathways involved in lung cancer pathology, as revealed by the KEGG pathway map, were tested on A549 and NCI-H226 human lung cells. Effects on viability, apoptosis, cell cycle, DNA damage, reactive oxygen species, and IL-8 secretion were determined after 24-h mycotoxin treatment at concentrations between 0.01 and 100 µM. Enniatin A, beauvericin and apicidin presented a similar response for all endpoints. Moreover, they were the most toxic compounds, with IC50 around 2.2 and 13.1 µM, and apoptosis induction in both cell lines. Enniatin B did not induce a complete decrease in cell viability and did not provoke apoptosis. Destruxin B increased caspase 3 activity and altered cell cycle at concentrations above 6.25 µM in A549 cells, suggesting apoptosis activation, although it did not reduce cell viability in either cell line. Finally, enniatins A and B, apicidin, beauvericin and destruxin B increased the release of the pro-inflammatory IL-8. The in vitro results of this work confirmed the prediction using in silico approaches as some hallmarks of cancer can be observed after treatment of lung cells with this mycotoxin cluster. The toxic effects observed in human lung cells confirmed that the presence of these emerging mycotoxins in agricultural products, food and feed might be hazardous for human health and that a link with lung cancer was further outlined for three of them.