Background The southern Amazon Basin in the Madre de Dios region of Peru has undergone rapid deforestation and habitat disruption, leading to an unknown zoonotic risk to the growing communities in the area. Methodology/Principal findings We surveyed the prevalence of rodent-borne Leptospira and Bartonella, as well as potential environmental sources of human exposure to Leptospira, in 4 communities along the Inter- Oceanic Highway in Madre de Dios. During the rainy and dry seasons of 2014-2015, we captured a total of 97 rodents representing 8 genera in areas that had experienced different degrees of habitat disturbance. Primarily by using 16S metagenomic sequencing, we found that most of the rodents (78%) tested positive for Bartonella, whereas 24% were positive for Leptospira; however, the patterns differed across seasons and the extent of habitat disruption. A high prevalence of Bartonella was identified in animals captured across both trapping seasons (72%-83%) and the relative abundance was correlated with increasing level of land disturbance. Leptospira-positive animals were more than twice as prevalent during the rainy season (37%) as during the dry season (14%). A seasonal fluctuation across the rainy, dry, and mid seasons was also apparent in environmental samples tested for Leptospira (range, 55%-89% of samples testing positive), and there was a high prevalence of this bacteria across all sites that were sampled in the communities. Conclusions/Significance These data indicate the need for increased awareness of rodent-borne disease and the potential for environmental spread along the communities in areas undergoing significant land-use change.