Abstract Background Species of the Tabanidae are potent vectors of human and animal diseases, but they have not been thoroughly investigated to date. In Senegal (West Africa), little information is available on these dipterans. Our objective in this study was to investigate Senegalese tabanids and their diversity by using molecular and proteomics approaches, as well as their associated pathogens. Methods A total of 171 female tabanids were collected, including 143 from Casamance and 28 from Niokolo-Koba. The samples were identified morphologically by PCR sequencing and by MALDI-TOF MS, and PCR analysis was employed for pathogen detection and blood-meal characterization. Results The morphological identification revealed four species concordantly with the molecular identification: Atylotus fuscipes (79.5%), Tabanus guineensis (16.4%), Chrysops distinctipennis (3.5%) and Tabanus taeniola (0.6%) (not identified by PCR). The molecular investigation of pathogens revealed the presence of Trypanosoma theileri (6.6%), Leishmania donovani (6.6%), Setaria digitata (1.5%), Rickettsia spp. (5.1%) and Anaplasmataceae bacteria (0.7%) in A. fuscipes. Tabanus guineensis was positive for L. donovani (35.7%), S. digitata (3.6%) and Anaplasmataceae (17.8%). Leishmania donovani has been detected in 50% of C. distinctipennis specimens and the only T. taeniola specimen. No Piroplasmida, Mansonella spp. or Coxeilla burnetii DNA was detected. In addition to humans (96.43%), Chlorocebus sabeus, a non-human primate, has been identified as a host of (3.57%) analysed tabanids. MALDI-TOF MS enabled us to correctly identify all tabanid species that had good quality spectra and to create a database for future identification. Conclusions Tabanids in Senegal could be vectors of several pathogens threatening animal and public health. To fully characterize these dipterans, it is therefore necessary that researchers in entomology and infectiology employ molecular characterization and mass spectrometric techniques such as MALDI-TOF MS to analyse these dipterans in Senegal and West Africa.