This study examined the in vitro potential of bioconjugated quantum dots (QDs) as photosensitizers for photodynamic therapy (PDT). According to our previous approaches using photosensitizers, folic acid appears to be an optimal targeting ligand for selective delivery of attached therapeutic agents to cancer tissues. We synthesized hydrophilic near infrared emitting CdTe(S)-type QDs conjugated with folic acid using different spacers. Photodynamic efficiency of QDs conjugated or not with folic acid was evaluated on KB cells, acting as a positive control due to their overexpression of FR-α, and HT-29 cells lacking FR-α, as negative control. A design of experiments was suggested as a rational solution to evaluate the impacts of each experimental factor (QD type and concentration, light fluence and excitation wavelength, time of contact before irradiation and cell phenotype). We demonstrated that, for concentrations lower than 10 nM, QDs displayed practically no cytotoxic effect without light exposure for both cell lines. Whereas QDs at 2.1 nM displayed a weak photodynamic activity, a concentration of 8 nM significantly enhanced the photodynamic efficiency characterized by a light dose-dependent response. A statistically significant difference in photodynamic efficiency between KB and HT-29 cells was evidenced in the case of folic acid-conjugated QDs. Optimal conditions led to an enhanced photocytotoxicity response, allowing us to validate the ability of QDs to generate a photodynamic effect and of folic acid-conjugated QDs for targeted PDT.