Mosquitoes are arthropods that represent a real public health problem in Africa. Morphology and molecular biology techniques are usually used to identify different mosquito species. In recent years, an innovative tool, matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), has been used to identify many arthropods quickly and at low cost, where equipment is available. We evaluated the ability of MALDI-TOF MS to identify mosquitoes collected in Senegal and stored for several months in silica gel, and to determine the origin of their blood meal. A total of 582 mosquitoes were collected and analysed. We obtained 329/582 (56.52%) MALDI-TOF MS good-quality spectra from mosquito legs and 123/157 (78.34%) good-quality spectra from engorged abdomens. We updated our home-made MALDI-TOF MS arthropod spectra database by adding 23 spectra of five mosquito species from Senegal that had been identified morphologically and molecularly. These included legs from Anopheles gambiae, Anopheles arabiensis, Anopheles cf. rivulorum, Culex nebulosus, Anopheles funestus, and three spectra from abdomens engorged with human blood. Having updated the database, all mosquitoes tested by MALDI-TOF MS were identified with scores greater than or equal to 1.7 as An. gambiae (n = 64), Anopheles coluzzii (n = 12), An. arabiensis (n = 1), An. funestus (n = 7), An. cf rivulorum (n = 1), Lutzia tigripes (n = 3), Cx. nebulosus (n = 211), Culex quinquefasciatus (n = 2), Culex duttoni (n = 1), Culex perfescus (n = 1), Culex tritaeniorhynchus (n = 1), and Aedes aegypti (n = 2). Blood meal identification by MALDI-TOF MS revealed that mosquitoes had fed on the blood of humans (n = 97), cows (n = 6), dogs (n = 2), goats (n = 1), sheep (n = 1), and bats (n = 1). Mixed meals were also detected. These results confirm that MALDI-TOF MS is a promising technique for identifying mosquitoes and the origin of their blood meal.