The aim of this study was to perform a molecular survey and determine the genetic diversity of Bartonella spp. (Rhizobiales: Bartonellaceae) and Rickettsia spp. (Rickettsiales: Rickettsiaceae) in cat fleas (Siphonaptera: Pulicidae) from Southern Chile. Fleas (n = 251) were collected from 150 cats in Valdivia city and identified using morphological keys. Fleas belonging to the same cat were pooled (two to seven fleas per pool). DNA was purified from individual (n = 92) and pooled (n = 58) fleas and submitted to conventional polymerase chain reaction assays targeting Bartonella spp. (gltA) and Rickettsia spp. (ompA). Selected positive samples were sequenced for Bartonella gltA (n = 19), Rickettsia ompA (n = 14), and Rickettsia gltA (n = 11) for speciation, phylogenetic, and diversity analyses. All fleas (n = 251) were identified as Ctenocephalides felis felis (Bouché) (Siphonaptera: Pulicidae). Bartonella and Rickettsia occurrences in C. felis felis were 39.3% (59/150) and 76.6% (115/150), respectively. From sequenced Bartonella spp., 47.3% (9/19) were identified as Bartonella clarridgeiae, 42.1% (8/19) as Bartonella henselae, 5.3% (1/19) as Bartonella koehlerae, and 5.3% (1/19) as Bartonella sp. Rickettsia felis was the only Rickettsiaceae species identified in both ompA (14/14) and gltA (11/11) products. B. henselae and B. clarridgeiae presented five genotypes. R. felis ompA sequences presented seven genotypes. On the other hand, R. felis gltA sequences showed only one genotype. Bartonella spp. and R. felis are described for the first time in C. felis felis fleas from Southern Chile, highlighting the importance of these vectors as a source of zoonotic agents.