Objective The aim of the study was to evaluate the effect of doxycycline‐ and dexamethasone‐doped collagen membranes on the proliferation and differentiation of osteoblasts. Background Collagen barrier membranes are frequently used to promote bone regeneration and to boost this biological activity their functionalization with antibacterial and immunomodulatory substances has been suggested. Methods The design included commercially available collagen membranes doped with doxycycline (Dox‐Col‐M) or dexamethasone (Dex‐Col‐M), as well as undoped membranes (Col‐M) as controls, which were placed in contact with cultured MG63 osteoblast‐like cells (ATCC). Cell proliferation was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium (MTT) assay and differentiation by measuring the alkaline phosphatase (ALP) activity using spectrophotometry. Real‐time quantitative polymerase chain reaction was used to study the expression of the genes: Runx‐2, OSX, ALP, OSC, OPG, RANKL, Col‐I, BMP‐2, BMP‐7, TGF‐β1, VEGF, TGF‐βR1, TGF‐βR2, and TGF‐βR3. Scanning electron microscopy was used to study osteoblast morphology. Data were assessed using one‐way analysis of variance or Kruskal–Wallis tests, once their distribution normality was assessed by Kolmogorov–Smirnov tests (p > .05). Bonferroni for multiple comparisons were carried out (p < .05). Results Osteoblast proliferation was significantly enhanced in the functionalized membranes as follows: (Col‐M < Dex‐Col‐M < Dox‐Col‐M). ALP activity was significantly higher on cultured osteoblasts on Dox‐Col‐M. Runx‐2, OSX, ALP, OSC, BMP‐2, BMP‐7, TGF‐β1, VEGF, TGF‐βR1, TGF‐βR2, and TGF‐βR3 were overexpressed, and RANKL was down‐regulated in osteoblasts cultured on Dox‐Col‐M. The osteoblasts cultured in contact with the functionalized membranes demonstrated an elongated spindle‐shaped morphology. Conclusion The functionalization of collagen membranes with Dox promoted an increase in the proliferation and differentiation of osteoblasts.