Many conductive scaffolds were fabricated to mimic the topography of the cardiac microenvironment in vitro in order to improve the performance of engineered cardiac tissues. This study fabricated aligned poly(lactic-co-glycolic acid)/multiwalled carbon nanotube fibers by electrospinning. The Young's modulus and conductivity were significantly greater on poly(lactic-co-glycolic acid) fibers with 3% multiwalled carbon nanotubes compared to others. Topographical cues and conductivity were applied to investigate the combined effect on cardiomyocyte behavior. Neonatal rat cardiomyocytes cultured on the conductive fibers maintained their viability, induced cell elongation, and enhanced sarcomeric alpha-actinin and troponin I production in cardiomyocytes. The results indicate that poly(lactic-co-glycolic acid)/multiwalled carbon nanotube composite fibers have great potential for cardiac tissue engineering.