Combinations of growth factors synergistically enhance tissue regeneration, but typically require sequential, rather than co‐delivery from biomaterials for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings can deliver multiple factors without loss of activity; however, sequential delivery from PEM has been limited due to interlayer diffusion that results in co‐delivery of the factors. This study shows that addition of a biomimetic calcium phosphate (bCaP) barrier layer to a PEM coating effectively prevents interlayer diffusion and enables sequential delivery of two different biomolecules via direct cell access. A simulated body fluid method was used to deposit a layer of bCaP followed by 30 bilayers of PEM made with poly‐l‐Lysine (+) and poly l‐Glutamic acid (−) (bCaP‐PEM). Measurements of MC3T3‐E1 proliferation and viability over time on bCaP‐PEM were used to demonstrate the sequential delivery kinetics of a proliferative factor fibroblast growth factor‐2 (FGF‐2) followed by a cytotoxic factor (antimycin A, AntiA). FGF‐2 and AntiA both retained their bioactivity within bCaP‐PEM, yet no release of FGF‐2 or AntiA from bCaP‐PEM was observed when cells were absent indicating a cell‐mediated, local delivery process. This coating technique is useful for a variety of applications that would benefit from highly localized, sequential delivery of multiple biomolecules governed by cell initiated degradation that avoids off‐target effects associated with diffusion‐based release. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1500–1509, 2017.