Epidemiological surveillance is fundamental to optimize disease prevention and control strategies in aquaculture. Therefore, a disease surveillance program was implemented in 2023 with the highest representativeness of the tilapia farming industry in the South, Southeast, Midwest, Center-West and Northeast region of Brazil. Fish were collected from different farming systems (hatchery/hatchery tank, net tanks, among others) and production stages (fry, juveniles and adults). The program was based on the identification of viruses and bacteria by real-time qPCR directly in tissue samples and bacteriological culture with MALDI-TOF identification. In addition, antibiogram and minimum inhibitory concentration (MIC) assays were performed for all bacterial isolates obtained. Of the total 7200 qPCR analyses performed in 2023, Lactococcus spp. was the most prevalent pathogen (22%), followed by Streptococcus agatactiae (20%) and ISKNV (18%). From bacteriological cultures, 3285 bacterial isolates were obtained and identified as Aeromonas hydrophila (21.6%), S. agalactiae (21.3%), L. garvieae (14.3%), Edwarsiella tarda (11.3%), A. veronii (7.6%) and Francisella orientalis (5.6%). Of the total ISKNV positive cases, A. hydrophila was isolated in 44%, S. agalactiae in 24%, E. tarda in 15%, L. garvieae in 14% and F. orientalis in 2%, confirming a high prevalence rate of co-infection under field conditions. This epidemiological surveillance program is unprecedented in tilapia farming in Brazil and has helped to guide the adoption of prevention and control measures in the different production systems.