The aim of the study was to evaluate exposure of equids to rickettsial agents (Rickettsia rickettsii, Rickettsia parkeri, ‘Candidatus Rickettsia amblyommii’, Rickettsia rhipicephali, and Rickettsia bellii) and rickettsial infection in ticks of a Pantanal region of Brazil. Sera of 547 equids (500 horses and 47 donkeys) were evaluated by indirect immunofluorescence assay. In total, 665 adults and 106 nymphal pools of Amblyomma cajennense F. sensu lato, 10 Dermacentor nitens Neumann ticks, and 88 larval pools of Amblyomma sp. were tested by polymerase chain reaction (PCR). Overall, 337 (61.6%) equids were reactive (titer ≥64) to at least one antigen of Rickettsia spp. The prevalence values for Rickettsia were 66%, and the highest endpoint titers were observed for ‘Ca. R. amblyommii’. By PCR 3 (0.45%) A. cajennense s.l. females were positive for ‘Ca. R. amblyommii’. Minimum infection rates of 0.75% for nymphs and 0.34% for larvae were calculated. Positive samples of ticks have had a fragment of the 16S mitochondrial rRNA gene sequenced and sequences showed 99% identity to Amblyomma sculptum Berlese. This study reports a wide exposure of equids to Rickettsia agents, and PCR evidence of infection with ‘Ca. R. amblyommii’, for the first time, in A. sculptum.