Graphene based nanomaterials are promising candidates for cardiac tissue engineering due to the excellent electrical and mechanical properties and the robust surface chemistry. This research was designed to investigate the physicochemical and biological effects of increasing concentration of reduced graphene oxide (rGO) coating on collagen (Col) scaffolds as well as their antibacterial properties. Enhanced GO coating content to 400 μg/ml and its reduction showed improvement of HUVECs viability, however, following reduction of more GO concentration, decreased cell viability was observed. Compared with the Col counterpart, electroactive containing rGO scaffolds upregulated cardiac gene expression involve in electrical coupling (Cx43), muscle contraction and relaxation (troponin-T) and cytoskeleton alignment (actinin-4) after 7 days even without external electrical stimulation. rGO coating significantly improved mechanical properties and the electroactivity of the Col scaffolds reaching to 1100 ± 31 kPa and 4 × 10 ± 1.20 S/m for GO concentration of 800 μg/ml, respectively. Also, the antibacterial properties of Col-rGO-400 scaffolds against Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes were confirmed by culture and FESEM observation. Taken together, the results indicated that rGO coating presents promising properties to Col scaffolds providing a desirable micro environment for cardiomyocytes coupling and gene upregulation as well as antibacterial activities for cardiac patch application.