Aging is known to involve alterations in the composition and organization of the extracellular matrix, which have an impact on heart function. However, there is not a comprehensive description of how collagen characteristics vary with age in the human left ventricle (LV) and its impact on electrophysiological properties. Here, we quantified the amount and spatial organization of collagen from human LV second harmonic generation (SHG) microscopy images of middle-age and elderly individuals. The results were input to in silico models of human LV tissues and numerical simulations were conducted to characterize the effects on electrical conduction and repolarization. Results from SHG image processing showed an increase in the amount of collagen and in its clustering in LV tissues with age. The increase in the amount of fibrosis induced a clear decrease in conduction velocity (CV), whereas increased clustering did not impact CV in our simulated tissues. In terms of ventricular repolarization, we observed a remarkable reduction in action potential duration (APD) as the percentage of fibrosis increased and a slighter reduction with increasing clustering. Importantly, more clustered fibrosis had a major effect on the enhancement of spatial APD dispersion, which was, however, diminished with increased fibrosis percentage. As a conclusion, both the amount and spatial organization of fibrosis in human LV tissues have a relevant role in electrophysiological properties.