The outbreak of lactococcosis affecting rainbow trout, weighing 70 - 120 g, occurred at a trout aquaculture facility in Central-West Serbia during July, 2018. This outbreak lasted for three weeks, and cumulative mortality attributed to the disease was around 40%. The diseased fish showed erratic swimming, dark discoloration and exophthalmia, with the cumulative mortality of around 40%. A pure Gram-positive cocci isolates were obtained from the eye and kidney samples. Based on colony morphology, phenotypic and biochemical characteristics, the isolated bacterium was presumably identified as Lactococcus garvieae. Using the BBL CRYSTAL GP ID system, the isolate was identified as L. garvieae and the identity of the isolate was confirmed with MALDI-TOF Mass Spectrometry. Blast analysis of 16S rRNA sequence of our isolate had a 99.4 to 99.6% similarity to the L. garvieae strains previously isolated from diseased fish. The antibacterial activity of 15 antimicrobials against L. garvieae was evaluated using disc diffusion. In this paper, we report the first case of lactococcosis in rainbow trout in Serbia, isolation and characterization of causative agent, Lactococcus garvieae from diseased rainbow trout.