Add info This study aimed to identify proteins interacting with Fha in Vibrio cholerae. A Strep-tagged Fha fusion protein (Fha-Strep) was expressed in Vibrio cholerae, and associated protein complexes were enriched using Strep-Tactin affinity purification following cell lysis. As a negative control, an sfGFP-Strep fusion protein was expressed and processed in parallel. The enriched protein samples were subjected to tryptic digestion and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a timsTOF Pro mass spectrometer operated in dia-PASEF mode. This approach enabled comprehensive identification and quantification of Fha-associated proteins. Three biological replicates were included for each group to ensure reproducibility. The resulting dataset provides a resource for understanding the interaction network of Fha and its potential role in the Type VI Secretion System (T6SS) in Vibrio cholerae.