Rodent-borne bartonellae have been identified as human pathogens. Little is known about Bartonella infections in woodrat hosts and their fleas and how woodrat-flea associations may affect the dynamics of Bartonella infections. We collected blood samples and fleas from two species of woodrats (Neotoma micropus and N. albigula) from Santa Fe County, NM, from 2002–2005. The most predominant flea species were Orchopeas sexdentatus and O. neotomae. Bartonella prevalence in woodrats was 64% overall, with a lower prevalence occurring in the pre-reproductive period compared to the early and late reproductive periods. A negative correlation between Bartonella prevalence in N. micropus and weight of N. micropus was observed. Flea load in Neotoma species was highest in the early reproductive period compared to the pre- and late reproductive periods and was higher in N. micropus compared to N. albigula. Bartonella prevalence in fleas was highest in the early reproductive period and lowest in the late reproductive period, and it was higher in fleas collected from N. micropus than in fleas collected from N. albigula. Abundance of O. sexdentatus was significantly higher in N. micropus compared to N. albigula, and abundance of O. sexdentatus and O. neotomae was highest in the early reproductive period. No direct correlations were found either between Bartonella prevalence in woodrats and in fleas or between Bartonella prevalence in woodrats and flea loads. Out of 25 partially characterized Bartonella isolates from Neotoma woodrats, 24 belonged to one genogroup based on sequencing of the gltA gene.