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Rickettsioses, often underreported, pose public health challenges. <i>Rickettsia asembonensis</i> is a potential emerging pathogen that was previously detected in humans, animals, and a variety of arthropods. While its pathogenicity in humans remains unclear, it poses a potential public health threat. Here, we present an extended epidemiological, diagnostic, and genetic analysis of the information provided in a preliminary report on the investigation of rickettsiae in Peru. In particular, we report the detection of <i>R. asembonensis</i> in blood specimens collected from four human patients with an acute undifferentiated fever of a seven- to nine-day duration, all of whom tested negative for other vector-borne pathogens. Additionally, we describe the replicative capacity of the <i>R. asembonensis</i> isolates in cell cultures.