The discovery of giant viruses, with capsids as large as some bacteria, megabase-range genomes and a variety of traits typically found only in cellular organisms, was one of the most remarkable breakthroughs in biology. Until recently, most of our knowledge of giant viruses came from ~100 species-level isolates for which genome sequences were available. However, these isolates were primarily derived from laboratory-based co-cultivation with few cultured protists and algae and, thus, did not reflect the true diversity of giant viruses. Although virus co-cultures enabled valuable insights into giant virus biology, many questions regarding their origin, evolution and ecological importance remain unanswered. With advances in sequencing technologies and bioinformatics, our understanding of giant viruses has drastically expanded. In this Review, we summarize our understanding of giant virus diversity and biology based on viral isolates as laboratory cultivation has enabled extensive insights into viral morphology and infection strategies. We then explore how cultivation-independent approaches have heightened our understanding of the coding potential and diversity of the Nucleocytoviricota. We discuss how metagenomics has revolutionized our perspective of giant viruses by revealing their distribution across our planet’s biomes, where they impact the biology and ecology of a wide range of eukaryotic hosts and ultimately affect global nutrient cycles.