Fleas are carriers for many largely understudied zoonotic, endemic, emerging, and re-emerging infectious disease agents, but little is known about their prevalence and role as a vector in Africa. The aim of this study was to determine the diversity of fleas and the prevalence of infectious agents in them collected from human dwellings in western Kenya. A total of 306 fleas were collected using light traps from 33 human dwellings; 170 (55.56%) were identified as
Ctenocephalides
spp., 121 (39.54%) as
Echidnophaga gallinacea
, 13 (4.25%) as
Pulex irritans
, and 2 (0.65%) as
Xenopsylla cheopis
. Of the 306 individual fleas tested, 168 (54.9%) tested positive for rickettsial DNA by a genus-specific quantitative real-time PCR (qPCR) assay based on the 17-kDa antigen gene. Species-specific qPCR assays and sequencing revealed presence of
Rickettsia asembonensis
in 166 (54.2%) and
Rickettsia felis
in 2 (0.7%) fleas.
Borrelia burgdorferi
, normally known to be carried by ticks, was detected in four (1.3%) flea DNA preparations. We found no evidence of
Yersinia pestis
,
Bartonella
spp., or
Orientia
spp. Not only were
Ctenocephalides
spp. the most predominant flea species in the human dwellings, but also almost all of them were harboring
R. asembonensis.