BackgroundTicks are obligate bloodsucking parasites responsible for significant economic losses and concerns with human and animal health, mainly due to the transmission of pathogens. Entomopathogenic fungi have been intensively studied as an alternative strategy for tick control that can be used in combination with synthetic acaricides in the integrated management of ticks. Here, we investigated how the gut bacterial community ofRhipicephalus microplusis shaped afterMetarhizium anisopliaetreatment and how the tick susceptibility to the fungus is affected after disrupting gut bacterial microbiota.MethodsPartially engorged tick females were artificially fed with pure bovine blood or blood plus tetracycline. Two other groups received the same diet and were topically treated withM. anisopliae. The guts were dissected, and the genomic DNA was extracted 3 days after the treatment; the V3–V4 variable region of the bacterial 16S rRNA gene was amplified.ResultsThe gut of ticks that received no antibiotic but were treated withM. anisopliaeexhibited lower bacterial diversity and a higher occurrence ofCoxiellaspecies. The Simpson diversity index and Pielou equability coefficient were higher in the gut bacterial community whenR. micropluswere fed with tetracycline and fungus-treated. Ticks from fungus-treated groups (with or without tetracycline) exhibited lower survival than untreated females. Previous feeding of ticks with the antibiotic did not change their susceptibility to the fungus.Ehrlichiaspp. were not detected in the gueated groups.ConclusionsThese findings suggest that myco-acaricidal action would not be impacted if the calf hosting these ticks is under antibiotic therapy. Moreover, the hypothesis that entomopathogenic fungi can affect the bacterial community in the gut ofR. microplusengorged females is endorsed by the fact that ticks exposed toM. anisopliaeexhibited a dramatic reduction in bacterial diversity. This is the first report of an entomopathogenic fungus affecting the tick gut microbiota.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05790-5.