Giant viruses significantly regulate the ecological dynamics of diverse ecosystems. Although metagenomics has expanded our understanding of their diversity and ecological roles played in marine environments, little is known about giant viruses of freshwater ecosystems. Most previous studies have employed short-read sequencing and therefore resulted in fragmented genomes, hampering accurate assessment of genetic diversity. We sought to bridge this knowledge gap and overcome previous technical limitations. We subjected spatiotemporal (2 depths × 12 months) samples from Lake Biwa to metagenome-assembled genome reconstruction enhanced by long-read metagenomics. This yielded 294 giant virus metagenome-assembled genomes. Of these, 285 included previously unknown species in five orders of nucleocytoviruses and the first representatives of freshwater mirusviruses, which exhibited marked divergence between freshwater- and marine-derived lineages. Notably, 42 (14.3%) genomes were composed of single contigs with completeness values >90%, demonstrating the good performance of our long-read metagenomic assembly. Giant viruses were partitioned across water depths, with most species specific to either the sunlit epilimnion or the dark hypolimnion. Epilimnion-specific members tended to be opportunistic and to exhibit short and intense abundance peaks, in line with the fact that they regulate the surface algal blooms. During the spring bloom, mirusviruses and members of three nucleocytovirus families were among the most abundant giant viruses. In contrast, hypolimnion-specific ones including algaviruses and mirusviruses were typically more persistent in the hypolimnion throughout the water-stratified period, suggesting that they infect hosts specific to the hypolimnion and play previously unexplored ecological roles in dark water-specific microbial ecosystems.