A quantitative proteomics approach investigated the natural co-infected Labeo rohita kidney. A kidney proteome of L. rohita was utilized to understand the in-depth knowledge of rohu for disease mechanisms. Comparative proteome analysis was performed for our earlier submitted proteome of control and Edwardsiella tarda infected kidney using label free quantitative proteomics. High-resolution mass spectrometry was used for data using label-free quantification. Label-free proteomic analysis showed pathogenicity mechanisms related to apoptosis, lysosome, metabolic pathway, immune system and cellular response to stress in the kidney of co-infected rohu. The study specifies that co-infection for the first time in the winter season was capable of causing pathological changes in L. rohita. The initial proteome analysis of naturally co-infected rohu kidneys identified novel proteins that could aid in developing strategies for combating multiple pathogens and enhancing climate resilience in aquaculture.