A diversity of Borrelia burgdorferi sensu lato (Johnson, Schmid, Hyde, Steigerwalt & Brenner) (Spirochaetales: Spirochaetaceae) genomospecies, including the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), have been identified in the western United States. However, enzootic transmission of B. burgdorferi s.l. in small mammals and ticks is poorly characterized throughout much of the region. Here we report prevalence of B. burgdorferi s.l. in small mammal and tick communities in the understudied region of southern California. We found B. burgdorferi s.l. in 1.5% of Ixodes species ticks and 3.6% of small mammals. Infection was uncommon (∼0.3%) in Ixodes pacificus Cooley and Kohls (Acari: Ixodidae), the primary vector of the Lyme disease agent to humans in western North America, but a diversity of spirochetes—including Borrelia bissettiae, Borrelia californiensis, Borrelia americana, and B. burgdorferi s.s.—were identified circulating in Ixodes species ticks and their small mammal hosts. Infection with B. burgdorferi s.l. is more common in coastal habitats, where a greater diversity of Ixodes species ticks are found feeding on small mammal hosts (four species when compared with only I. pacificus in other sampled habitats). This provides some preliminary evidence that in southern California, wetter coastal areas might be more favorable for enzootic transmission than hotter and drier climates. Infection patterns confirm that human transmission risk of B. burgdorferi s.s. is low in this region. However, given evidence for local maintenance of B. burgdorferi s.l., more studies of enzootic transmission may be warranted, particularly in understudied regions where the tick vector of B. burgdorferi s.s. occurs.