The biological activity of chemical retraction/displacement agents in surrounding periodontal tissues is of unquestionable importance, but the activity of these agents has not been completely elucidated. In the present study, we aimed to evaluate the in vitro effects of vasoconstrictive retraction agents on primary human gingival fibroblasts (HGFs). A total of six commercial adrenergic solutions (0.05 and 0.01% HCl-epinephrine, two based on 0.05% HCl-tetrahydrozoline, 0.05% HCl-oxymetazoline, and 10% HCl-phenylephrine) and three experimental gel formulations (EG-1, EG-2, and EG-3) were used to treat primary HGFs. The biological effect of the retraction treatment on the expression of collagen types I and III was detected by performing immunocytochemical analysis. The generation of reactive oxygen species triggered by the retraction agents were evaluated by using the dichlorofluorescein (DCF) fluorescent probe. The effect of retraction agents on the expression of fibronectin was visualized by confocal laser scanning microscopy. According to the results, experimental retraction gels did not limit the expression of collagen types I and III. EG-3 even induced the synthesis of both types of collagen. The DCF assay indicated oxidative stress similar to the control cells for most of the selected retraction agents. Experimental gels did not cause degradation of the cellular shape and morphology of the primary HGFs. The proposed experimental retraction gels in the present study demonstrated higher biocompatibility with primary HGFs, suggesting their use as clinical vasoconstrictive agents for the application of gingival retraction with minimal damage to periodontal tissues.