Objective Evaluation of the anti‐proliferative mechanisms in HeLa cells cultures by cyclodipeptides from Pseudomonas aeruginosa PAO1. Methods Cyclodipeptides from P. aeruginosa PAO1 (PAO1‐CDPs) cultures were obtained by solvent extraction according to Ortiz‐Castro et al. (2011). Cell viability of HeLa cells was determined by the MTT method and apoptosis using Annexin‐V according to Vázquez‐Rivera et al. (2014). Mitochondrial membrane potential determination and real‐time quantification of superoxide was carried out by FACS according to Gómez et al. 2014. Immunodetection was conduced using human antibodies: anti‐Akt, anti‐Akt‐phosphorylated (Ser473), anti‐p70S6 kinase, anti‐phosphorylated‐p70S6 kinase (Thr 389); developed using Goat anti‐Rabbit IgG HRP‐conjugate. Abstract Pseudomonas aeruginosa PAO1 produces the cyclodipeptides cyclo(L‐Pro‐L‐Tyr), cyclo(L‐Pro‐L‐Phe), and cyclo(L‐Pro‐L‐Val), whose effects have been implicated in inhibition of proliferation in human tumor cell lines. Our purpose was to elucidate the anti‐proliferative mechanisms caused by the PAO1‐CDPs in HeLa cells cultures. The results indicate that the PAO1‐CDPs both individually as in mixture, inhibited HeLa cell proliferation by arresting the cell cycle at the G0–G1 transition. The PAO1‐CDPs mixture promoted cell death in HeLa line by a dose‐dependent manner, showing half‐lethal dose (LD50) of 60 μM and inducing apoptosis at 0.6 μM. Moreover, PAO1‐CDPs showed a higher pro‐apoptotic activity (~10 fold) than their synthetic analogs. Subsequently, the mitochondrial membrane potential was arrested by the PAO1‐CDPs and induced apoptosis by caspase‐9‐dependent pathway. The anti‐proliferative mechanism in HeLa cells involves inhibition of phosphorylation of both the Akt‐S473 and S6k‐T389 protein kinases, showing a cyclic behavior of their phosphorylation in a time and concentration‐dependent fashion. Conclusions Our findings indicate that the PI3K/Akt/mTOR/S6k signaling pathway blocking is involved in the anti‐proliferative effect of the PAO1‐CDPs.