High-content phenotypic screening, encompassing both drug and genomic screenings, constitute an invaluable research and discovery tool, which has been extensively explored by the industry and academia for various aims, including target discovery and validation, drug discovery and development, drug repurposing/repositioning, and mechanistic studies. In particular the application of functional genomic screenings using genome-wide RNAi, CRISPR and microRNA libraries in complex cell-based assays offers a unique opportunity to systematically probe the function of classical drug targets as well as of otherwise undruggable targets, enabling the investigation of molecular networks underlying complex biological and disease-relevant processes. Through the application of large-scale image-based screening approaches, we have been identifying and characterizing novel cellular factors (proteins and microRNAs) and drugs implicated in different biological contexts and pathologies, including cardiovascular disorders, infection and tumorigenesis. Overall, these studies have uncovered unexpected molecular players and pathways, revealing novel opportunities for therapeutic intervention.