Background. Periradicular surgery is the last treatment option for teeth with persistent periradicular endodontic lesions. This study aimed to assess the adhesion of fibroblasts to root dentin conditioned with ethylenediaminetetraacetic acid (EDTA), MTAD, and QMix. Methods. Twelve dentin discs were fabricated of 6 human single-rooted teeth. Fibroblasts were isolated from the periodontal ligament (PDL) of a premolar tooth. The teeth were healthy and freshly extracted from the socket. The samples were divided into four groups for surface conditioning with (I) EDTA, (II) MTAD, (III) QMix, and the control group. Fibroblasts were cultured on conditioned dentin discs at 37°C, 95% air, and 5% CO2 for 4 hours and then rinsed with PBS three times to eliminate unattached cells from the surface. The mean counts of attached cells were calculated using a Neubauer chamber. Also, the attachment of fibroblasts was evaluated using scanning electron microscopy (SEM). Results. The mean counts of fibroblasts attached to root dentin in EDTA, QMix, MTAD, and control groups were 303±46, 243±41, 213±33, and 347±38, respectively. No significant difference was noted in the number of fibroblasts attached between MTAD, EDTA, and QMix and the control group (P>0.05). Under SEM, the fibroblasts were flat and spindle-shaped, with cytoplasmic processes covering the untreated dentin surface. In the experimental groups, the cells were rounder with fewer processes. All the three groups showed weaker adhesion to dentin compared to the control (untreated dentin) group. Conclusion. Under the limitations of this study, it was concluded that treating the dentin surface with EDTA, MTAD, or QMIX might not be an effective way to improve the adhesion of human PDL fibroblasts.